060 100

IDENTIFICATION BY PHYSICAL MEANS OF
ORGANIC MOIETIES OF CONJUGATES PRODUCED

FROM CARBARYL BY TOBACCO CELLS IN
SUSPENSION CULTURE
RAYMOND K. LOCKE~, JO-YUN T. CHEN z, JOSEPH N. DAMICO2'3
LAURENCE R. DUSOLDz, and JAMES A. SPHONz
1Division of Toxicology and 2Division of Chemistry and Physics,
Food and Drug Administration, U.S. Department of Health,
Education, and Welfare) Washington, D.C. 20204
Carbaryl (1-naphthyl methylcarbamate), labeled with ~4C in the C~-naphthyl, car-

bonyl, or N-methyl position, was introduced into the culture medium of tobacco cells
in suspension culture. Following incubation, cells were homogenized in water, cen-
trifuged, and supernatants hydrolyzed with /3-glucosidase or HCI. Organic moieties
(moieties) were characterized by two-dimensional thin-layer chromatography (TLC),
and many were subsequently identified by infrared and mass spectrometry. On the
basis of the data obtained with 14C~-naphthyl-labeled carbaryl, it appeared that 18.4%
of the total characterized metabolites represented unconjugated N-CH~OH- carbaryl
[l-naphthyl N-(hydroxymethyl)carbamate], excreted by the cells into the culture
medium. The metabolites found in the cells primarily consisted of conjugates of
l-naphthol (73.6% of the total characterized metabolites) and N-CHzOH-carbaryl
(2.5%). Conjugates of 7-hydroxycarbaryl (7-hydroxy-l-naphthyl methylcarbamate),
4-hydroxycarbaryl (4-hydroxy-l-naphthyl methylcarbamate), and 5-hydroxycarbaryl
(5-hydroxy-l-naphthyl methylcarbamate) were also detected in small amounts. Of five
unknown 14C1-naphthyl-labeled carbaryl metabolites, three were tentatively charac-
terized as: O-l-naphthylcholesterol (cholest-5-en-3/3-yl-l-naphthol; 3.0%); an uncon-
jugated hydroxylated 1,4-dihydro-l,4-epiperoxynaphthalene (1.4%); and an acid-
labile, fl-glucosidase-resistant conjugate of a cis-dihydrodiol of 1-naphthol (0.3%;
other than the trans-5,6-dihydrodiol). The cholesterol derivative may represent a new
"detoxification mechanism" in plants; the epiperoxide may help to elucidate plant
oxidation mechanisms. A new TLC procedure was developed which successfully
separated the acetate d e r i v a t i v e o f N - h y d r o x y c a r b a r y l ( l - n a p h t h y l N-
hydroxy-N-methylcarbamate) from 12 other common moieties of carbaryl metabolites
and their acetate derivatives. A new two-dimensional TLC system was developed for
the separation of underivatized N-hydroxycarbaryl from 14 other moieties of carbaryl
metabolites; two additional two-dimensional TLC systems were uti'lized for moiety
separations. With these TLC p r o c e d u r e s , no c o n j u g a t e d or unconjugated
N-hydroxycarbaryl could be detected in any tobacco cell culture fraction after incuba-
tion of cells in medium containing radiolabeled carbaryl. Authentic ~4C~-naphthyl-
labeled N-CH~OH-carbaryl was shown to be converted to desmethylcarbaryl (1-
naphthylcarbamate) (97%) and l-naphthol (3%) by 0. IN HCI hydrolysis.
Earlier reports from this laboratory ( L o c k e et al. 1971, L o c k e 1972a) have

suggested that up to 16% o f the total m e t a b o l i t e s p r o d u c e d from 14C1-naphthyl-
ZDeceased.
Archives of Environmental Contamination 60

and Toxicology Vol. 4, 60-100 (1976)
01976 by Springer-Verlag New York Inc.
Physical Identification of Organic Moieties 61
labeled carbaryl (l-naphthyl methylcarbamate) by tobacco cells in suspension cul-

ture might represent N-O-conjugates of N-hydroxycarbaryl (l-naphthyl
N-hydroxy-N-methylcarbamate). This suggestion was based solely upon co-
chromatography with authentic standards of organic moieties (hereafter referred to
simply as moieties) liberated from tobacco cell conjugates by acid hydrolysis,
utilizing one-dimensional thin-layer chromatography (TLC).
Since carbaryl is a widely used insecticide, it was important to investigate by

physical methods the production of N-hydroxycarbaryl conjugates from carbaryl by
tobacco cells. Such carbaryl conjugates in the food supply might pose a serious
toxicological hazard for man, with regard to the liberation of free N-hydroxylated
derivatives of carbaryl following ingestion. Baskakov and Zemskaya (1959)
suggested that plants are capable of the N-hydroxylation of isopropyl N-phenyl-
carbamate, a carbamate herbicide. Although the biological effects of N-hydroxylated
derivatives of carbaryl are as yet unknown, the N-hydroxylated derivatives of 1- and
2-naphthylamine (Radomski and Brill 1970; Belman et al. 1968, Mayer 1972,
Mukai and Troll 1969, Perez and Radomski 1965) and 2-acetylaminofluorene
(Cramer et al. 1960, Miller et al. 1960) have been shown to be mutagenic or
carcinogenic in a variety of biological systems.
Although the metabolism of carbaryl by higher plants has been less extensively
studied than its metabolism by mammalian and insect systems, excellent reviews of
the earlier plant metabolism data are currently available (Kuhr 1970, Fukuto 1972,
Casida and Lykken 1969). More comprehensive studies have recently appeared
utilizing TLC (Andrawes et al. 1972) and mass and ultraviolet spectrometry
(Mumma et al. 1971) for the determination of moiety structures. In no case have
N-O-conjugates of N-hydroxycarbaryl been reported as plant metabolites of car-
baryl. However, structural identifications of plant conjugate moieties by physical
means have been few in the earlier studies, and the TLC systems employed in the
tentative assignments of structure have frequently been deficient with regard to the
separation of N-hydroxycarbaryl from other carbaryl metabolite moieties (Locke
1972b).
Plant cell culture techniques have not been widely used in investigations of
pesticide metabolism, although previous studies from this (Locke et al. 1971, Locke
and Baron 1972) and other (Feung et al. 1971, 1973) laboratories have shown that
these techniques are useful for preliminary studies of pesticide degradation by higher
plants. The purpose of the present study was to structurally identify by physical
methods the moieties derived from carbaryl metabolites produced by tobacco cells in
suspension culture, and to relate these in vitro metabolites with the in vivo metabo-
lites reported for higher plants. Particular attention was given to establishing the
presence or absence of conjugates of N-hydroxycarbaryl.
Materials and methods

Liquid scintillation counting. Fluor A, a scintillation phosphor previously de-
scribed (Baron and Locke 1970), was used in the radioassay of particulate and
62 R . K . Locke et al.
high-protein samples, except that the concentration of thixotropic gel (Cab-O-Sil)

was increased from 33.3 to 50.0 g per liter. As noted, water was added to certain
samples contained in Fluor A (one ml of water per 20 ml of phosphor) to aid in the
formation of a thick, stable gel. Fluor B, consisting of Fluor A with thi• gel
omitted, was utilized in the radioassay of nonparticulate and low-protein samples.
The instrumentation and the procedures used for both organic and aqueous samples
were previously described (Locke et al. 1971).
Extraction procedure. To one volume of a given culture fraction, one volume of

methylene chloride was added. After extraction, the phases were separated and the
organic phase was dried over anhydrous Na2SO4. In all cases, both phases were
radioassayed. This procedure resulted in the quantitative recovery of radioactivity in
the organic phase, with the extraction of either culture medium or untreated cell
supernatant (SN) to which radiolabeled carbaryl had been added.
The extraction of enzymic or acid hydrolysates of SN by this procedure fre-

quently resulted in the formation of emulsions. In such cases, up to one additional
volume of methylene chloride was added to the original mixture, and the sample was
re-extracted. Residual emulsions, if present, were broken by centrifugation.
Acid hydrolysis. Culture fractions were titrated to pH 1.0 with HC1 and hyd-
rolyzed for one hr (except as noted) at 100~ on a steambath. The hydrolysates were
then extracted, with prior neutralization to pH 7.0 with NaOH if indicated.
Thin-layer chromatography. A. S o l v e n t systems and standards. Noncommercial

100-/z TLC plates prepared from Silica Gel G-F254 (E. Merck) were used for TLC.
The solvent systems and the sources of solvents and chromatographic standards have
been previously described (Locke 1972b). The following additional solvent systems
were used: System XV, chloroform-acetonitrile 3:1 (v/v); and System XVI,
cyclohexane-ethyl acetate 9:1 (v/v). All of these additional solvents were
"distilled-in-glass" reagents (Burdick and Jackson Laboratories, Inc.) with the
exception of the cyclohexane (99.5%; Phillips Petroleum Co.). The following addi-
tional standards were obtained from Union Carbide Chemicals Co.: desmethyl-
carbaryl (1-naphthyl carbamate); 7-hydroxycarbaryl (7-hydroxy-l-naphthyl methyl-
carbamate); N-acetylcarbaryl (l-naphthyl N-acetyl-N-methylcarbamate); N-acetyl-
desmethyl carbaryl (1-naphthyl N-acetylcarbamate); and the naphthalene- 1,3; - 1,4;
- 1,5; - 1,6; and - 1,7-diols. The standards of 5,6-dihydro-5,6-dihydroxy- 1-naphthyl
methylcarbamate and 5,6-dihydro-5,6-dihydroxy-l-naphthol utilized in the present
and previous (Locke 1972b, Locke et al. 1971) studies were both the trans isomers.
Naphthalene-2,3-diol was obtained from Aldrich Chemical Co., Inc. Scopoletin
(6-methoxy-7-hydroxycoumarin) was obtained from K&K Laboratories, Inc.
B. One-dimensional analytical TLC ( 1 D - A - T L C ) . For 1D-A-TLC, organic ex-

tracts were spotted on the TLC plate, with development for 15 cm above the origin.
Radioactivity was located by scraping 1.0- or 0.5-cm sections of the silica gel and
counting each section in ten ml of Fluor A. Authentic standards (ten /zg) were
chromatographed simultaneously with unknowns in all cases. Compounds were

detected by viewing with UV light as previously described (Locke 1972b).
C. One-dimensional preparative TLC (1D-P-TLC). For the preparative isolation

of moieties for spectometry, organic extracts were streaked on 100-g TLC plates
and subjected to repetitive 1D-P-TLC in several solvent systems (15 cm solvent
fronts). The solvents utilized to extract moieties from the silica gel, as well as the
precautions taken to avoid contaminants which might interfere with spectrometric
analyses, have been previously described (Locke and Mayer 1974, Chen and Dority
1970).
D. Two-dimensional analytical TLC (2D-A-TLC). For 2D-A-TLC, organic ex-

tracts containing radiolabeled moieties were added to a mixture of nonradioactive
authentic standards (20 /~g of each standard) in acetone. The mixture was then
spotted 2.5 cm from the bottom and left side of a 100-/~ TLC plate. The plate was
developed in the first direction for 15 cm above the origin, removed from the tank,
briefly dried with cold air, and developed similarly in the second direction. Solvent
was evaporated, and the standards were made visible with short- and long-
wavelength UV light. Standards were outlined with microspatula prickings, and a
tracing was made of the TLC plate for use in analytical calculations. Distinct spots
o f silica gel corresponding to known standards were scraped from the plate and
radioassayed in ten ml of Fluor A.
When all spots corresponding to known standards had been removed from the
TLC plate, the remaining silica gel was divided into 25 squares, 4 x 4 cm, with
microspatula prickings. The silica gel contained in each square was scraped and
radioassayed with added water in 20 ml of Fluor A. Radioactivity corresponding to
compounds other than the standards could thus be located on the TLC plate and
quantitated. Radioactivity corresponding to a known standard was expressed as a
percentage of the total radioactivity recovered from the plate.
E. Two-dimensional preparative TLC (2D-P-TLC). Certain organic extracts were

subjected to 2D-TLC, as described in Section D, but in the absence of added
standards. The moieties were located by absorbance or fluorescence with U V light,
and the extracts were analyzed by infrared and mass spectrometry.
Thin-layer chromatographic procedure for the detection and separation of

N-hydroxycarbaryl from other moieties of carbaryl metabolites by formation of
the acetate derivative. Commercial 250-~ Silica Gel G-F254 TLC plates (E. Merck)
were utilized in this procedure. Organic extracts were applied to a TLC plate and
overspotted with ten /zl of acetic anhydri.de; authentic standards were also applied
and treated similarly. The plate was incubated (without development) for 15 min at
room temperature in a tank saturated with acetic anhydride vapor.
The TLC plate was placed in an oven at 105~ for five min to remove the acetic
anhydride from the silica gel. After the plate had cooled, standards and extracts not
to be acetylated were applied. The plate was subjected to 1 D - A - T L C in S y s t e m

X V I , and c o m p o u n d s were made visible with U V light. R a d i o c h r o m a t o g r a m s o f
organic extracts were o b t a i n e d as described in Section B. The authentic standards
subjected to this p r o c e d u r e are e n u m e r a t e d in Table I.
Table I. Thin-Layer chromatographic procedure for the detection of

N-hydroxycarbaryl as its acetate derivative a
Compound b Rff
N-Hydroxycarbaryl 0.03
N-Hydroxycarbaryl + AA a 0.19
1-Naphthol 0.28
l-Naphthol + AA 0.28; 0.48
N-Acetylcarbaryl 0.33
N-Acetylcarbaryl + AA 0.33
Carbaryl 0.07
Carbaryl + AA 0.08
N-CHzOH-Carbaryl 0.00
N-CH2OH-Carbaryl + AA 0.00
4-OH-Carbaryl 0.00
4-OH-Carbaryl + AA 0.01
5-OH-Carbaryl 0.01
7-OH-Carbaryl 0.00
N-Acetyldesmethylcarbaryl 0.05
N-Acetyldesmethylcarbaryl + AA 0.05
Desmethylcarbaryl 0.03
Desmethylcarbaryl + AA 0.03
trans-5,6-diH-5 ,6-diOH-Carbaryl 0.00
trans-5,6-diH-5,6-diOH-Carbaryl + AA 0.00
trans-5,6-diH-5,6-diOH- 1-Naphthol 0.00
trans-5,6-diH-5,6-diOH- l-Naphthol + AA 0.00
1,3-Naphthalenediol 0.03
1,3-Naphthalenediol + AA 0.03; 0.07; 0.20
1,4-Naphthalenediol + AA 0.40
1,5-Naphthalenediol + AA 0.08; 0. [ l; 0.19
1,7- Naphthalenediol 0.03
aprocedure described in "Materials and methods."

bChemical names are given in "Materials and methods."
eThe Rf values presented are averages of several independent determinations.
dlndicates treatment of the standard with acetic anhydride (AA).
Infrared (IR) spectrometry. All IR spectra, except those of metabolite-derived

and standard l-naphthyl acetate, were obtained with micro KBr discs (Chen 1965,
Chen and Dority 1970), using a Perkin-Elmer IR grating spectrophotometer, Model
621, equipped with a 6X dual condensing accessory. The IR spectra of 1-naphthyl
acetate samples were obtained in carbon disulfide, using three-mm micro KBr sealed
liquid cells.
Mass spectrometry. Low-resolution mass spectra were generally obtained with

an Atlas CH-4B mass spectrometer. Certain low- and high-resolution spectra were
obtained with an AEI MS-30 mass spectrometer (Shrader Analytical and Consulting
Laboratories, Inc., Detroit, Mich.). In both cases, a direct probe inlet system and an
ionizing current of 70 eV were utilized.
Ultraviolet (UV) spectrometry. UV spectra were obtained with a Beckman Acta

V dual-beam recording spectrophotometer, using one-ml cuvettes of one cm light
path. Spectra were determined in ethanol or methylene chloride against the approp-
riate reference solvent. Additions of acids or bases to the sample cuvette were
concomitantly made to the reference cuvette.
Radiolabeled compounds and culture conditions used. In all metabolism

studies, the chemical concentration of the 14C-radiolabeled carbaryl in the culture
medium was nine ppm. The radiolabeled compounds were dissolved in methylene
chloride and added to sterile 250-ml Erlenmeyer flasks. The flasks were stoppered
(plastic foam plugs) and the solvent was allowed to evaporate. The radioactive
compounds utilized were t4Ct-naphthyl- [synthesized or Union Carbide Corpora-
tion; specific activity (SA) = 1,080 dpm//zg], N-t4CH.~- (Union Carbide Corpora-
tion, SA = 32,281 dpm//~g), ~4CO-labeled carbaryl (Nuclear-Chicago; SA = 396
dprn//.Lg). The stated specific activities were obtained by dilution of radiolabeled
compounds with nonradioactive carbaryl (commercial; Union Carbide Corporation;
recrystallized from ethanol). The chemical and radiometric purities of each labeled
carbaryl were verified before use by ID-A-TLC in System I.
A 30-ml portion of a ten-day suspension culture of tobacco cells and 100 ml of

liquid M-1D medium were added by aseptic techniques to each flask containing the
dry radiolabeled carbaryl. The flasks were placed on a gyrorotatory shaker (Model
" V " ; New Brunswick Scientific Co., Inc. ; approximately 200 rev/min), and incuba-
tion was continued for 14 days in the dark at 27~ To minimize contamination, the
cultures were incubated in a closed room equipped with UV lighting. The results of
incubating 14C,-naphthyl-labeled carbaryl for 12 days in cell-free culture medium
have already been reported (Locke et al. 1971).
The XD cell line of Nicotiana tabacum L. var. xanthi, the chemically defined
liquid M-1D culture medium, and the routine subculturing procedures used for
suspension cultures have been previously described (Filner 1965, Locke et al.
1971). Cells obtained from suspension cultures could be conveniently stored as
callus cultures on agar M-1D medium for a period of one month or more before
transfer to fresh agar medium. As needed, suspension cultures were obtained by the
addition of one g or more of callus tissue to 100 ml of liquid M-1D medium. In
contrast to the procedures followed by Filner (1965), both callus and suspension
cultures were cultivated in flasks stoppered with uncapped plastic foam_plugs (open
system). Media flasks were capped with aluminum foil during storage to prevent
evaporation.
To insure proper growth, suspension cultures of XD tobacco cells should initially

contain greater than 5 x 103 cells/ml (Filner 1965). In studies utilizing 14C~-
naphthyl-, N-t4CHz -, and t4CO-radiolabeled carbaryl, the number of cells added per
flask to initiate the cultures were the following: 5.0 x 106, 9.0 • 106, and 2.6 •
108 , respectively. Cell number was approximated by determinations o f the dry
weights of ceils, freed of medium by a water wash, contained in culture aliquots.
For tobacco cells cultured for ten days or longer, one mg of cell dry weight is
equivalent to approximately 6.7 x 104 fresh cells (Filner 1973).
Two series of experiments were conducted with 14C~-naphthyl-labeled carbaryl,

utilizing 30 cultures for each series. Six cultures were used for studies of the
metabolism of 14CO-labeled carbaryl. One culture was used in the investigation of
the metabolism of N-14CH3-1abeled carbaryl.
Sterility testing. Aliquots (one ml) of the culture media from representative
cultures were aseptically added to 100-ml portions of both thioglycollate medium
and soybean-casein digest medium (Sterility Test Media Formulations I and III,
respectively; U.S. Pharmacopeia 1970). The sterility test cultures were incubated for
seven days at either 37~ (thioglycollate) or 27~ (soybean-casein digest), and
contamination was judged by cloudiness with respect to controls. Tobacco cells will
not grow in these media.
Fractionation of suspension cultures. Following incubation, the cells were

allowed to settle, and the culture medium was decanted. The medium was then
filtered by vacuum and an aliquot was radioassayed in ten ml of Fluor B. The filter
paper (Schleicher & Schuell No. 597), containing decanted ceils, was cut into small
strips which were transferred to several vials and radioassayed with added water in
20 ml of Fluor A. Although Miracloth (Chicopee Mills, Inc., New York, N.Y.) can
be used to harvest cells directly from suspension cultures more easily (Filner 1965),
it was not utilized in the present study.
The cells were repeatedly washed with nonradioactive M-ID medium and cen-
trifuged at 700 x g for 20 min at 4~ until no significant radioactivity could be
detected in the final wash. Aliquots of each wash were radioassayed in Fluor B. The
washed cells were homogenized in water (about 1:1 w/v) at 0~ (on ice), using a
glass tissue homogenizer equipped with a motor-driven Teflon pestle. Aliquots of
the homogenate were taken with a wide-bore-tipped pipette (a pipette with a portion
of the tip removed) and radioassayed with added water in 20 ml o f Fluor A.
Radioactivity contained in the tobacco cell homogenate, together with the radioac-
tivity found in the filter paper used for filtration of the culture medium, was taken as
the total radioactivity incorporated into the tobacco ceils. No direct measurement
was made of 14CO2 evolution.
Fractionation of radioactivity incorporated into tobacco cells. The tobacco

cell homogenate was centrifuged at 700 x g for 20 rain at 4~ The supernatant (SN)
was decanted, and the cell debris pellet was repeatedly washed with water and
centrifuged under the same conditions, until no significant radioactivity appeared in
the final wash. Aliquots of the SN were radioassayed in Fluor A; aliquots of each
cell debris wash were radioassayed in Fluor B. The washed cell debris pellet was
resuspended in water and aliquots, obtained with a wide-bore-tipped pipette, were
radioassayed with added water in 20 ml of Fluor A. Radioactivity appearing in the
SN and cell debris wash fractions was taken to represent soluble metabolites, while
radioactivity appearing in the washed cell debris was taken to represent metabolites
bound or incorporated into cell debris components.
Investigation of metabolites contained in the culture medium. Filtered culture

media from tobacco cultures incubated with 14Cl-naphthyl- or N-14CHz-labeled
carbaryl were extracted. The organic extracts were concentrated by rotary evapora-
tion, and aliquots were subjected to 2D-A-TLC (System I followed by System XV).
The remainder of the organic extract of the medium containing N-~4CHa-labeled

metabolites was subjected to ID-P-TLC in System XV. The UV-absorbing material
co-chromatographing with a standard ofN-CHzOH-carbaryl (R~ = 0.48) was eluted,
re-chromatographed in System XV, and the extract was analyzed by IR spectromet-
ry.
Investigation of metabolites incorporated into tobacco cells. A. fl-Glucosidase

treatment of t4Cl-naphthyl-labeled cell metabolites. In one series of experiments
utilizing ~4C~-naphthyl-labeled carbaryl, an aliquot of the untreated SN was ex-
tracted. A portion of the organic extract was concentrated (Nz) and subjected to
2D-A-TLC (System I followed by System XV). In addition, an aliquot of the extract
was subjected to 1D-P-TLC in System VIII, and the radioactivity exhibiting a Re
value of 0.33 (moiety M - l ) was eluted with ethyl acetate and re-chromatographed in
System VIII. The ethyl acetate extract was then analyzed by Fourier-transform
NMR, IR, and mass spectrometry.
To establish the lability of radiolabeled conjugates contained in the SN to en-

zymic hydrolysis, an aliquot was incubated with a mixture of fl-glucosidase and aryl
sulfatase. To ten ml of 0.3M (total citrate) citric acid-sodium citrate buffer at pH
5.2, two ml of SN (22,600 dpm) and 18.ml of distilled water were added. To the
resultant mixture (0.1M in total citrate) were added 1.0 mg of/3-glucosidase (Sigma
Chemical Co.; emulsin from almonds) and 0.1 mg of aryl sulfatase (Sigma Chemical
Co.; Type II from limpets). Toluene (0.035 ml) was added to retard bacterial
growth, and the mixture was incubated for five hr at 37~ in a stoppered Erlenmeyer
flask with constant shaking.
A control was prepared by incubating an aliquot of SN under identical conditions,

except for the absence of enzymes. After incubation, the samples were extracted.
The activities of the /3-glucosidase and aryl sulfatase enzyme preparations were
established using substrates and methodology previously described. (Locke and
Baron 1972).
The remainder of the SN was treated with/3-glucosidase only. To 346 ml of SN,

containing 4 • 106 dpm, 173 ml of 0.3M citrate buffer at pH 5.2 was added, and to
the resulting mixture (0.1M in total citrate) 10.0 mg of/3-glucosidase was added
with constant stirring. The mixture was divided into 26-ml portions and incubated in
stoppered Erlenmeyer flasks for 16.5 hr at 37~ with constant shaking. Toluene
(0.035 ml) was added to each flask to retard bacterial growth. After incubation, the
enzymic hydrolysate was radioassayed, adjusted to pH 7.0 with NaOH, and ex-
tracted.
An aliquot of the organic phase from the extraction of SN incubated with

/3-glucosidase only was subjected to 2D-A-TLC (System I followed by System XV).
The remainder of this organic extract was subjected to ID-P-TLC on 2 TLC plates in
System XIV. The major moieties isolated from these TLC plates, and the solvent
systems used for their further sequential TLC purification prior to IR spectrometry,
were the following: 1) N-CH2OH-carbaryl [System XIV (Rf = 0.20); System IX (Rr
= 0.35); System I followed by System XV (See Figure I-A)]; 2) l-naphthol
[System XIV (R, = 0.58); System I (Rf = 0.73)]; and 3) carbaryl [System XIV (R,
= 0.41); System I followed by System XV (See Figure l-A)].
In addition, the radioactivity on the two preparative TLC plates (System XIV)
from 0 to 2.3 cm above the origin was eluted with acetone and subjected to
1D-P-TLC in System VIII, together with standards of phenol, benzoic acid, t r a n s -
5,6-dihydro-5,6-dihydroxy-l-naphthyl methylcarbamate, and t r a n s - 5 , 6 - d i h y d r o -
5,6-dihydroxy-l-naphthol. A UV-absorbing moiety, designated M-2, co-chromato-
graphed with benzoic acid (R~ = 0.29; streaked from the origin). The silica gel
containing M-2 was sequentially extracted with acetone, ethanol, methanol, and
water. The UV spectrum of M-2 was determined with the ethanol extract.
The water phase from the extraction of the/3-glucosidase hydrolysate of SN was

then acid hydrolyzed (1.5 hr), and the hydrolysate was extracted, following
neutralization with NaOH. The organic phase was concentrated b y rotary evapora-
tion, and an aliquot was subjected to 2D-A-TLC (System I followed by System
XV).
The remainder of the organic extract of the acid hydrolysate was streaked on two
TLC plates and subjected to 1D-P-TLC in System VIII with known standards. The
following areas of silica gel were scraped from the TLC plates, extracted with
acetone and then with methanol, and the dried (N2) combined extracts for each
sample were analyzed by high-resolution mass spectrometry: 1) 5.7 to 7.7 cm
(moiety M-3) and 2) 8.0 to 10.5 cm (moiety M-4).
B if Solvent front 1 (System II) A C
t4~# ~ I [ 1112~ 1311p~ ~ t Solvent front 1 (System I) 14 r~
R"
I N-OH ~ ~ 111
R"
gz
I !k2 4 O
9
Origin ~ = 1 CM Origin ~'~ = 1 CM 0--, = 1 CM
,-I
Fig. 1. Two-dimensional analytical thin-layer chromatography of authentic standards of carbaryl metabolite moieties, under conditions
described in "Materials and methods," and in the following solvent systems: A. System X1V followed by System XIII; B. System I! 2.
followed by System XV; and C. System I followed by System XV. The designations used for moieties, and the colors which they
displayed upon heating of the thin-layer plate with a stream of warm air, are the following: N-OH = l-naphthyl N-hydroxy-N-
methylcarbamate (colorless); 1 = trans-5,6-dihydro-5,6-dihydroxy-l-naphthyl methylcarbamate (colorless); 2 = trans-5,6-dihydro-5,6-
dihydroxy-l-naphthol (tan); 3 = l-naphthyl N-(hydroxymethyl)carbamate (colorless); 4 = 7-hydroxy-l-naphthyl methylcarbamate
(pink); 5 = 4-hydroxy-l-naphthyl methylcarbamate (orange); 6 = 5-hydroxy-l-naphthyl methylcarbamate (yellow); 7 = l-naphthyl
carbamate (colorless); 8 = l-naphthyl methylcarbamate (colorless); 9 = naphthalene-l,3-dioi (orange); 10 = naphthalene-l,7-diol
(purple-grey); 11 = naphthalene-l,6-diol (green-grey); 12 = naphthalene-1,5-diol (green-purple-grey); 13 = l-naphthol (grey-tan); and
14 = naphthalene-l,4-diol (yellow).
B. Acid hydrolysis of ~4C1-naphthyl-labeled cell metabolites. In the second series

of experiments utilizing 14C~-naphthyl-labeled carbaryl, the washed cell debris was
resuspended in water and subjected to acid hydrolysis. The hydrolysate was ex-
tracted. No further studies were conducted with the cell debris fraction.
An aliquot of non-hydrolyzed SN was extracted. The remainder of the SN was

acid hydrolyzed and the hydrolysate was extracted. An aliquot of the organic phase
was then subjected to the TLC procedure for the detection of N-hydroxycarbaryl as
its acetate derivative. The remainder of the extract was subjected to 1D-P-TLC on
two plates in System XIV with known standards. Distinct UV-absorbing or fluores-
cent bands were scraped from the TLC plates and eluted with acetone.
Material co-chromatographing with a carbaryl standard (Rf = 0.42) was further

purified by 1D-P-TLC in System I (Rt = 0.34), and the organic extract was analyzed
by mass and IR spectrometry. Radioactivity co-chromatographing with a standard of
l-naphthol (Rt = 0.48) was eluted and evaporated to dryness. The acetate derivative
was formed by adding one ml of acetic anhydride to the dry extract and incubating at
room temperature for one hr. The excess acetic anhydride was evaporated (Nz).
Standard l-naphthyl acetate was prepared by similar treatment of authentic
1-naphthol. The radiolabeled derivative was then subjected to ID-P-TLC in System
XVI, together with standards of 1-naphthol and 1-naphthyl acetate, l-Naphthol (Rf
= 0.25) was well separated from 1-naphthyl acetate (Rt = 0.41) in System XVI.
The radioactivity co-chromatographing with the 1-naphthyl acetate standard was
analyzed by IR and mass spectrometry.
Material contained in the organic extract of the acid hydrolysate and co-
chromatographing on the preparative TLC plates with a standard of N-hydroxy-
carbaryl (Rf = 0.32) was subsequently purified by ID-P-TLC in System XIII (Rf =
0.50) and System II (Rt = 0.59). The organic extract was then analyzed by mass and
IR spectrometry. A fluorescent material (Rf = 0.22), initially thought to be
radiolabeled, was detected after preparative TLC of the organic extract of the acid
hydrolysate in System XIV. This fluorescent compound was designated F-1 and was
purified by 1D-P-TLC in System VIII (Rf = 0.95) and in System X (Rf = 0.71).
Although the final extract of F-I contained no significant radioactivity, aliquots
were analyzed by mass, UV, and IR spectrometry.
The silica gel was scraped from one cm below to two cm above the origins of the
two plates used for preparative ID-P-TLC of the organic extract of the acid hydroly-
sate, and was combined. The eluate was purified by ID-P-TLC in System VIII.
Radioactivity with a Rf value of 0.30 was designated M-5, and was further purified
by ID-P-TLC in System V (Re = 0.20) and in System VIII (Rf = 0.40). Insufficient
material was recovered for spectrometric analyses.
C. Acid Hydrolysis of 14CO-labeled cell metabolites. The SN obtained from

tobacco cells incubated with 14CO-labeled carbaryl was acid hydrolyzed, and the
hydrolysate was extracted. Aliquots of the organic phase were subjected to ID-A-
TLC in System XIII and System XIV and to the TLC procedure described for the
detection of N-hydroxycarbaryl as its acetate derivative.
To the remainder of the organic phase, 160 /zg of authentic nonradioactive

N-hydroxycarbaryl was added. The mixture was then subjected to 1D-P-TLC in
System XIV with known standards. The UV-absorbing material representing the
added N-hydroxycarbaryl standard (Rr = 0.45) was scraped from the TLC plate and
the silica gel was eluted with ethyl acetate. Aliquots of the extract were subjected to
2D-A-TLC (System XIV followed by System XIII, and System I followed by
System XV) and to the TLC procedure for the detection of N-hydroxycarbaryl as its
acetate derivative.
It was noted that radioactivity in the organic extract to which authentic

N-hydroxycarbaryl had been added co-chromatographed on the preparative TLC
plate with a standard of carbaryl (Rr = 0.71). This material was eluted, and aliquots
of the extract were subjected to 2D-A-TLC (System XIV followed by System XIII,
and System I followed by System XV).
. D. Acid hydrolysis of N-14CHa-labeled cell metabolites. The entire SN obtained

from tobacco cells incubated with N-14CHa-labeled carbaryl was extracted. The
organic phase was concentrated (Nz), and aliquots were subjected to 2D-A-TLC
(System II followed by System XV, and System I followed by System XV).
The water phase from the extraction of the untreated SN was acid hydrotyzed,
and the hydrolysate was extracted. To aliquots of the organic phase, 20 p.g of
authentic nonradioactive N-hydroxycarbaryl was added and the aliquots were sub-
jected to 1D-A-TLC in either System XIII or System XIV. The added startdard was
located by viewing under UV light, and the radiochromatograms were developed as
previously described.
To the remainder of the organic phase obtained from extraction of the acid
hydrolysate were added 20 ~g of each of the following nonradioactive compounds:
N-hydroxycarbaryl, carbaryl, and 4-hydroxycarbaryl. The mixture was subjected to
1D-P-TLC in System XIV, and the UV-absorbing materials corresponding to the
added standards were eluted from the silica gel with acetone.
The acetone extracts of materials co-chromatographing with the added standards

ofN-hydroxycarbaryl (Rf = 0.55) and carbaryl (Rf = 0.81) were concentrated (N2).
An aliquot of each extract was then subjected to 2D-A-TLC (System XIV followed
by System XIII). The acetone extract obtained from the silica gel remaining on the
preparative TLC plate, after the removal of the added standards, was also subjected
to 2D-A-TLC (System II followed by System XV).
Demonstration of the conversion of authentic N-CH2OH-carbaryl to

desmethylcarbaryl by acid hydrolysis. The use of acid hydrolysis for the liberation
of moieties from carbaryl metabolites causes structural alterations in certain cases.
Carbaryl itself is stable to 0.1N or 1.0N HC1 (Locke et al. 1971). On the other hand,
N-hydroxycarbaryl is significantly labile to acid at HC1 concentrations greater than
0 . I N (Locke 1972b). Wiggins et al. (1970) have shown that trans-5,6-dihydro-5,6-
dihydroxy-l-naphthyl methylcarbamate is converted to the 5-hydroxy-l-naphthyl
derivative by acid treatment. Qualitatively, these investigators also reported that
N-CH2OH-carbaryl was degraded by acid treatment to desmethylcarbaryl and
1-naphthol. It was therefore important to investigate in a quantitative manner the
stability of N-CH2OH-carbaryl to the conditions of acid hydrolysis employed in our
study.
x4CI-Naphthyl-labeled N-CHzOH-carbaryl, previously obtained by/3-glucosidase

treatment of conjugates produced by tobacco cells incubated with 14C1-naphthyl-
labeled carbaryl, was utilized in this experiment. This isolated t4Cl-naphthyl-
labeled material had an IR spectrum identical to that of authentic N-CHzOH-
carbaryl.
SN was freshly prepared by homogenizing untreated cells in water (1:1 w/v) as

previously described. To 15.5 ml of the SN, 200 /zl of an acetone solution of the
radiolabeled N-CH2OH-carbaryl (2,800 dpm; 2.8 /~g) was added. After mixing, an
aliquot of the SN was radioassayed.
The mixture was acid hydrolyzed and the hydrolysate was extracted twice with
20-ml portions of methylene chloride. The pooled organic phase was concentrated
(Nz) and the entire extract was subjected to 2D-A-TLC (System I followed by
System XV).
Natural products liberated from the SN by acid hydrolysis interfered with the
normal pattern of the added standards in the developed 2D-A-TLC chromatogram.
Because of similarities with the normal pattern, an area of silica gel suspected of
containing N-CHzOH-carbaryl, the ring-hydroxylated carbaryls, N-hydroxycarbaryl,
and desmethylcarbaryl was scraped from the TLC plate and eluted with acetone. The
acetone extract was radioassayed. The remaining UV-absorbing or fluorescent spots
were scraped and radioassayed. The remainder of the silica gel was divided into
squares and radioassayed as previously described.
The acetone extract of the silica gel area described was concentrated (N2) and
again subjected to 2D-A-TLC (System I followed by System XV). The resulting
chromatogram exhibited the normal pattern of added standards and was analyzed as
previously described.
Results
Thin-layer chromatographic detection of N-hydroxycarbaryl. A. Formation

Rf values for authentic
and 1D-TLC of the acetate derivative. Table [ gives the
standards of moieties of carbaryl metabolites obtained by 1D-TLC in System XVI,
with or without prior treatment with acetic anhydride. N-Hydroxycarbaryl was
quantitatively converted by treatment with acetic anydride to a single product,

shown by IR spectrometry to be its acetate derivative (l-naphthyl N-acetoxy-N-
methylcarbamate). Furthermore, the acetate derivative of N-hydroxycarbaryl could
be separated from all of the other compounds tested by the subsequent 1D-TLC,
except for products obtained by acetic anhydride treatment of the 1,3-, 1,5-, 1,6-,
and 1,7-naphthalenediols (Table I).
l-Naphthol was converted in good yield by the derivatization procedure to a

single product that was shown by IR spectrometry to be l-naphthyl acetate, although
the conversion was not quantitative. The subsequent 1D-TLC separated 1-naphthyl
acetate from all of the other compounds investigated, although 1,4-naphthalenediol
(with or without treatment with acetic anhydride) exhibited somewhat similar TLC
behavior (Table I). In addition, underivatized 1-naphthol was likewise separated
from all other compounds tested, with the exception ofN-acetylcarbaryl (1-naphthyl
N-acetyl-N-methylcarbamate) with or without treatment with acetic anhydride. The
heating procedure used to remove the acetic anhydride from the silica gel was shown
to be effective, since the acetate derivatives of 1-naphthol or N-hydroxycarbaryl
could not be detected when these standards were applied following heating.
The 1,3-, 1,5-, 1,6-, and 1,7-naphthalenediols all reacted with acetic anhydride
to form products with Rf values of approximately 0.11 and 0.19 after ID-TLC in
System XVI (Table I). These products probably represent the monoacetate(s) and
the diacetate, respectively, of each of these naphthalenediols. The two monoacetates
expected to be formed from each of these naphthalenediols, with the exception of
1,5-naphthalenediol (which would form only a single monoacetate), were apparently
not resolved by ID-TLC in System XVI. The presumed diacetates (Rf = 0.19)
co-chromatographed with the acetate of N-hydroxycarbaryl (Table I). 1,4-Naph-
thalenediol apparently did not react with acetic anhydride; furthermore, an IR
spectrum of the standard (not treated with acetic anhydride) was consistent with its
presumed structure and showed little decomposition of this standard to 1,4-naph-
thoquinone.
These data indicated that a sample suspected of containing the radiolabeled

N-hydroxycarbaryl moiety must first be subjected to a procedure which separates
this moiety from the naphthalenediols, before the formation of the acetate derivative
and subsequent ID-TLC in System XVI for the TLC confirmation of the presence of
the acetate derivative of N-hydroxycarbaryl.
The best method for the positive TLC identification of the radiolabeled
N-hydroxycarbaryl moiety in an organic extract proved to be 2D-A-TLC (System
XIV followed by System XIII), extraction of the radioactivity co-chromatographing
with the added nonradioactive standard of N-hydroxycarbaryl, and derivatization of
the extract with acetic anhydride followed by 1D-A-TLC in System XVI to de-
monstrate radioactivity co-chromatographing with the authentic acetate derivative of
N-hydroxycarbaryl. In this way, the N-hydroxycarbaryl moiety could be separated
from the 16 other moieties of carbaryl metabolites listed in Table I and their acetate
derivatives.
74 R.K. Locke et al.
The Rr values obtained from standards subjected to this newly developed TLC
procedure (Table I) were quite reproducible. Since N-hydroxycarbaryl exhibits
chromatographic behavior similar to that of carbaryl and/or 5-hydroxycarbaryl in
several TLC systems commonly used in studies of carbaryl metabotism (Locke
1972b), the derivatization and detection procedure presented here may well prove to
be a valuable tool in establishing the presence or absence of the 1-naphthyl
N-hydroxy-N-methylcarbamate moiety in future studies of carbaryl metabolism.
B. 2D-TLC ofunderivatized N-hydroxycarbaryl. The two solvent systems used by

Locke (1972b) for the 1D-TLC separation of N-hydroxycarbaryl from other common
carbaryl metabolite moieties were utilized in 2D-TLC. Figure 1-A shows a
chromatogram of a mixture of authentic standards obtained by 2D-TLC in these
systems (System XIV followed by System XIII). The Rr values for each compound
tested, obtained under the conditions described in "Materials and methods", may be
directly calculated from the data presented. This 2D-TLC system was advantageous
for the detection of N-hydroxycarbaryl, since it widely separated N-hydroxycarbaryl
and desmethylcarbaryl from all of the other carbaryl metabolite moieties tested, and
provided an adequate separation of these two compounds as well. On the other hand,
the ring-hydroxylated carbaryls and the two trans-dihydrodiol moieties were poorly
resolved by this system, and N-CH2OH-carbaryl co-chromatographed with
5-hydroxycarbaryl (Figure l-A).
A 2D-TLC system developed by Wiggins et al. (1970) provided a good separa-

tion of N-CH~OH-carbaryl and ring-hydroxylated carbaryls from each other and
from other moieties (System II followed by System XV; Figure l-B).
A modification of the 2D-TLC system developed by Wiggins et al. (1970),

provided an excellent separation of hydroxylated carbaryls and was routinely used
for this purpose (System I followed by System XV; Figure l-C).
Organic extracts of untreated culture medium or cell SN did not contain organo-
extractable natural products that interfered with the normal pattern of the added
nonradioactive standards in the 2D-TLC. This was also true of organic extracts of
]3-gtucosidase hydrolysates of tobacco cell SN, although natural products were
observed at other positions on the 2D-TLC chromatograms. Organic extracts of acid
hydrolysates of cell SN, however, contained large amounts of an unknown natural
product that chromatographed as a large spot partially covering both the N-CH.,OH-
carbaryl and 7-hydroxycarbaryl standards in the 2D-A-TLC of extracts in System I
followed by System XV (Figure l-C), or in System II followed by System XV
(Figure I-B). The positions of other standards were unchanged. As discussed later,
the N-CH2OH-carbaryl moiety does not survive the acid hydrolytic conditions
employed; therefore, in actuality, only the 7-hydroxycarbaryl moiety was partially
masked by the unknown natural product, No data on the behavior of this natural
product with 2D-TLC in System XIV followed by System XIII were obtained. Thus,
the only detectable interference by natural products with the pattern of added
standards in 2D-TLC chromatograms occurred solely with extracts of acid hydroly-
sates and affected only the 7-hydroxycarbaryl moiety.
Demonstration of the conversion of authentic N-CH2OH-carbaryl to des-

methylcarbaryl by acid hydrolysis. After acid hydrolysis of the ~C~-naphthyl-
labeled N-CH2OH-carbaryl standard in the presence of SN, all of the added radioac-
tivity could be recovered in the organic extract of the hydrolysate. When the organic
extract was subjected to 2D-A-TLC, the acetone extract of the silica gel area
described in "Materials and methods" contained 97% of the recovered radioactivi-
ty. None of the remaining UV-absorbing or fluorescent spots (representing natural
products or added standards) were radioactive, except for the spot corresponding to
a standard of 1-naphthol (3%). No radioactivity could be detected in the silica gel
remaining on the TLC plate after removal of the distinct spots and the silica gel area
described.
When the acetone extract of the scraped silica gel area was again subjected to
2D-A-TLC, the pattern of added standards was normal and all of the recovered
radioactivity was contained in the spot representing a standard of desmethylcarbaryl.
No radioactivity co-chromatographed with a standard of N-CH2OH-carbaryl or was
detected at any other position on the TLC plate.
Collectively, these data indicated that under the conditions of acid hydrolysis
used in our study, all liberated N-CH2OH-carbaryl would be converted to
desmethylcarbaryl (97%) and l-naphthol (3%), with the probable formation of
formaldehyde (Figure 2).
Metabolism of 14Cl-naphthyl-labeled carbaryl. A. Distribution of radioactivity

in culture fractions. After incubation of tobacco cell suspension cultures with
14Cx-naphthyl-labeled carbaryl, the added radioactivity was distributed among the
various culture fractions as shown in Table II. Table III shows the distribution of the
radioactivity recovered in fractions obtained by homogenizing washed tobacco cells.
B. Metabolites contained in the culture medium. When the culture medium was
extracted, 95% of the radioactivity was organoextractable. The extract was sub-
jected to 2D-A-TLC, and 97.6% of the recovered radioactivity corresponded to a
carbaryl standard, while 2.2% represented N-CHzOH-carbaryl. Thus, 1.459% of the
added carbaryl had been converted to N-CHzOH-carbaryl by the tobacco cells, and
i01 / CH20H O /H
O--C-N O-C-N OH
\ H 0.1 N HCI
1 hr; 100"C
100% 97% 3%
Fig. 2. Acid hydrolysis of authentic 14Ct-naphthyl-labeled N-CH.,OH-carbaryl in the presence

of tobacco cell supernatant as described in "Materials and methods." Products were iden-
tified by analytical two-dimensional thin-layer chromatography. Formaldehyde was not iso-
lated.
T a b l e II. Distribution of radioactivity in tobacco cell suspension cultures incubated

with 14C-radiolabeled carbaryl (percentage of added radioactivity)a
Added radioactivity
Washed Cell Culture Loss in ceils/Number
14C Position cells wash medium (14CO~.) of cells added b
C1-Naphthyl 30.2 14.5 55.3 0.0 6.0 x 10-6

Carbonyl 2.9 7.7 42.1 47.3 1.1 • 10-8
N-Methyl 13.6 22.4 64.0 0.0 1.5 x 10-8
aCulture conditions are described in "Materials and methods."

bSee "Materials and methods" for the number of cells added per flask in studies with each of
the radiolabeled carbaryls.
was apparently excreted unconjugated into the culture medium (Table IV). For
purposes of calculation, cell washes were considered diluted culture medium.
C. Metabolites contained in the cell debris. Only 1% of the radioactivity con-

tained in the cell debris fraction could be recovered in the organic phase after the
extraction of a 0. IN HC1 hydrolysate. The radioactivity contained in the cell debris
fraction was therefore not further investigated.
D. Metabolites contained in non-hydrolyzed SN. Only 2% of the radioactivity

contained in untreated SN was organoextractable. When an aliquot of the organic
extract was subjected to 2D-A-TLC, 66% of the recovered radioactivity corres-
ponded to a carbaryl standard, while 3% co-chromatographed with a standard of
N-CH~OH-carbaryl. Therefore, 0.012% of the added carbaryl had been converted to
unconjugated N-CH2OH-carbaryl contained in the SN and cell debris washes (Table
IV). When a portion of the extract was subjected to 1D-P-TLC in System VIII, 26%
of the radioactivity exhibited a Re value of 0.33 (M-l). The identity of M-1 is as yet
unknown. On the basis of Fourier-transform NMR, IR, and high-resolution mass
T a b l e I I I . Distribution of radioactivity incorporated into tobacco cells

following incubation with 14C-radiolabeled carbaryl (percentage of total
cellular radioactivity) a
Cell Cell debris Washed

1~C Position supernatant washes cell debris
C t-Naphthyl 52.7 14.7 32.6

Carbonyl 40.3 15.8 43.9
N-Methyl 71.1 18.6 10.3
aFractionation of the radioactivity incorporated into tobacco cells is described in

"'Materials and methods."
spectrometry, preliminary indications might suggest a hydroxylated 1,4-dihydro-

1,4-epiperoxynaphthalene as a possible structure. This speculation will be verified
by further studies on the chemical nature of M-I, which represented 0. 107% of the
added carbaryl (Table IV). For purposes of calculation, cell debris washes were
considered diluted SN.
E. Moieties liberated from tobacco cell supernatant (SN) by treatment with

fl-glucosidase. After SN was incubated with/3-glucosidase for 16.5 hr (l,345-fold
excess of enzyme), 20% of the radioactivity present in the hydrolysate could be
extracted into methylene chloride. When an aliquot of the SN was incubated for five
hr under essentially the same conditions with a mixture of/3-glucosidase (7,212-fold
excess) and aryl sulfatase (31-fold excess), 24% of the radioactivity present in the
hydrolysate was organoextractable. An aliquot of the SN incubated for five hr in the
absence of enzymes yielded 2% of the radioactivity in the organic phase after
extraction. The latter value agreed with that obtained in the organic phase after
extraction of untreated, non-incubated SN.
These data indicated that 18% of the radioactivity contained in the SN rep-
resented conjugates labile to fl-glucosidase, and that the hydrolysis of these conju-
gates had gone to completion during the 16.5-hr incubation period. In addition, the
mixture of aryl sulfatase and/3-glucosidase was only very slightly, if at all, more
effective in liberating radioactivity from the conjugates than was /3-glucosidase
alone. The recovery of added radioactivity from all enzymic hydrolysates was
quantitative.
With 2D-A-TLC of an aliquot of the organic extract of the /3-glucosidase hyd-

rolysate, 64.9% of the recovered radioactivity co-chromatographed with a standard
of l-naphthol, 5.0% with N-CH2OH-carbaryl, 4.3% with carbaryl, 0.6% with
7-hydroxycarbaryl, 0.3% with 4-hydroxycarbaryl, and 0.3% with 5-hydroxy-
carbaryl. The percentages of the added carbaryl represented by/3-glucosidase-labile
conjugates of each of these moieties, which were contained in the SN and debris
washes, are shown in Table IV. The figure given in Table IV for/3-glucosidase-
labile conjugates of N-CH2OH-carbaryl has been corrected for unconjugated
N-CH2OH-carbaryl contained in the SN and cell debris washes.
The remainder of the organic extract of the /3-glucosidase hydrolysate was sub-
jected to 1D-P-TLC. Radioactive moieties were purified by repetitive TLC and
identified by comparisons of their IR spectra with those of authentic standards.
When the IR or mass spectra of moieties corresponded to those already published for
authentic standards, the spectral reference (SR) only is given. The following
moieties of /3-glucosidase-labile conjugates were identified by IR spectrometry:
1-naphthol (SR: Pouchert 1970). N-CH,,OH-carbaryl (Figure 3-A), and carbaryl
(SR: Chen and Benson 1966).
An unknown carbaryl metabolite moiety, designated M-2, represented 0.059% of

the added carbaryl (Table IV). The conjugate(s) of this 14Cl-naphthyl-labeled moi-
T a b l e IV. Organic moieties recovered from tobacco cell cultures incubated with
laC l-naphthyl-labeled carbaryl a
% of total
% of added characterized
Moiety ~ Form radioactivity moieties
l-Naphthol Glucosidee'a 2.635 33.19

Conjugate(s)e,e 3.094 38.97
N-CH2OH-Carbaryl Unconjugatedg 1.459 18.38
Unconjugatedh 0.012 0.15
Glucosidee'd 0.191 2.41
Conjugate(s)e.f 0.008 0. i0
7-OH-Carbaryl Glucosidee'a 0.024 0.30
Conjugate(s)e'r 0.041 0.52
5-OH-Carbaryl Glucosidee'd 0.012 0115
Conjugate(s)e.t 0.012 0.15
4-OH Carbaryl Glucoside e,a 0.012 0.15
Conjugate(s)e.f 0.012 0.15
M- 1 (Unknown) Unconjugatedh 0.107 1.36
M-2 (Unknown) Glucosidee'a 0.059 0.74
M-3 (Dihydrodiol
of Naphthol) l Conjugate(s)~,t 0.021 0.26
M-5 (O-1-Naphthyl-
cholesterol) i Conjugate(s)ex 0.240 3.02
Total isolated metabolite moieties 7.939 I00.00
Carbaryl Unconjugatedg 64.720
Unconjugateda 0.175
Unconjugated~ 0.029
(Unconjugated)h,J (0.271)
Unknowns Water-solublesk 12.180
Water-solubles~ 3.490
Cell debris 9.900
Organoextractables
(by difference) 1.567
Total recovered radioactivity 100.000
aThe separation of cell culture fractions is described in "Materials and methods;"

metabolites contained in the cells were investigated utilizing hydrolysis by /3-glucosidase
and acid, as described in Section A.
bAbbreviations used for moieties are identified with chemical names in "Materials and
methods."
elndicates only that the moiety was liberated by hydrolysis with/~-glucosidase.
aObtained from the organic extract of the fl-g.lucosidase hydrolysate of cell supernatant and
cell debris washes.
elndicates that the moiety was not liberated by ~-glucosidase hydrolysis, but was labile to acid
hydrolysis.
fObtained from the organic extract of the acid hydrolysate of the water phase, obtained by
extraction of the /3-glucosidase hydrolysate of cell supernatant and cell debris washes.
ety was labile to fl-glucosidase, and the moiety had an Re value of 0.28 with
preparative 1D-TLC in System VIII. Standards of phenol (Rf = 0.98), trans-5,6-
d i h y d r o - 5 , 6 - d i h y d r o x y - l - n a p h t h y l methylcarbamate (Rf = 0.73), trans-5,6-
dihydro-5,6-dihydroxy-l-naphthol (Rf = 0.83), and benzoic acid (Rf = 0.29;
streaked from the origin) were chromatographed on the same TLC plate. When the
silica gel containing M-2 was sequentially eluted with acetone, ethanol, methanol,
and water, the percentages of the recovered activity found in the respective eluates
were 35, 7, 35, and 23%. Collectively, these data suggested that M-2 was a very
polar molecule.
The UV spectrum of an ethanol solution of M-2 exhibited a broad absorption

band centered at 271 nm and a small absorption band centered at 201 nm (Figure
4-A). After NaOH was added to both sample and reference cuvettes to an approxi-
mate concentration of 0.003N, an apparent shoulder appeared at 225 nm (Figure
4-A); the original spectrum could then be obtained by adding HC1 to a final
concentration of 0.03N, allowing for neutralization of the base. After an ethanol
solution of M-2 was saturated with sodium acetate, a sharp absorption band occurred
at 201 nm, in addition to the decreased maximum at 271 nm (Figure 4-B). The
spectrum of M-2 in an ethanolic 0. I N NaOH solution exhibited a sharp absorption
band at 220 nm, in addition to the unchanged maximum at 271 nm (Figure 4-B).
The UV spectrum of M-2 was different from those of all of the compounds listed
in Table I and from those of the following suspected moieties: benzoic acid,
cinnamic acid, cinnamyl alcohol, cinnamaldehyde, methyl cinnamate, o-hydroxy-
cinnamic acid, phthalic acid, phenol, naphthalene, 1,4-dihydronaphthalene, and
napthalene-2,3-diol. As mass and IR spectra of M-2 could not be obtained, this
moiety was not further investigated.
F. Moieties liberated by acid hydrolysis of the water phase obtained by extraction

of the fl-glucosidase hydrolysate. No loss of radioactivity due to hydrolysis occur-
red, and 25% of the radioactivity contained in the hydrolysate was organo-
extractable. With 2D-A-TLC of an aliquot of the organic extract, 76.2% of the
~Obtained from the organic extract of untreated culture medium and cell washes.
hObtained from the organic.extract of untreated cell supernatant and cell debris washes.
iTentative assignment of structure.
qncluded only to demonstrate that the carbaryl obtained from cell supernatant and cell debris
washes, following hydrolysis by /3-glucosidase or acid, represented only unconjugated
carbaryl; this value is not included in the sum.
kRadioactivity remaining in the water phase following the final extraction of cell supernatant
and cell debris washes treated with/3-glucosidase and subsequently with acid, as described
in Section A.
IRadioactivity remaining in the water phase following extraction of untreated culture medium
and cell washes.
80 R. K. Locke et al.
Wavelength (microns)
2.5 3 4 5 6 7 8 9 10 12 15 20 304050
100 I i i i
80
8= 60
1
E 40
I Wl
20
0 A
'I1
4000 3500 3000 2500 2000 1800 1600 1400 1200 1000 800 600 400 200
Wavenumber (CM - 1 )
2.5 3 4 5 6 7 8 9 10 12 15 20 30 40 50
100
80
'l_ I I ~L I I I I
v
~ 60 \/r Io.I tlr
g 4o
II I I i IJ llw ll
~ 20
0 !8
4000 3500 3000 2500 2000 1800 1600 1400 1200 1000 800 600 400 200
2.5 3 4 5 6 7 8 9 10 12 15 20 304050
100 ~ _
, {r -~,.
80
: 60 v "|
E 40 !
c
~- 20
C
0
4000 3500 3000 2500 2000 1800 1600 1400 1200 lOuU 800 600 400 200
Wavenumber (CM-1)
Fig. 3. Infrared spectrum: of A. N-CH._,OH-carbaryl liberated by treatment with/3-glucosidase

from conjugates produced from x4C~-naphthyl-labeled carbaryl and contained in the tobacco
cell supernatant and cell debris washes; B. authentic N-CH~OH-carbaryl; and C. N-CH~OH-
carbaryl produced by tobacco cells from N-14CH:3-1abeled carbaryl and present in the uncon-
jugated form in the culture medium and cell washes. Spectra were obtained as described in
"Materials and methods."
recovered radioactivity co-chromatographed with a standard of l-naphthol, 1.0%

with 7-hydroxycarbaryl, 0.3% with 4-hydroxycarbaryl, and 0.2% with desmethyt-
carbaryl. The percentages of the added carbaryl represented by acid-labile conju-
gates (not labile to fl-glucosidase) of each of these moieties are shown in Table IV.
Desmethylcarbaryl was taken to represent the moiety resulting from acid hydrolysis
1.0
Aj
0.9 !
!
0.8 i
.~ 0.7
C
--i
c5 0.6
o l' i / r
c
0.5
0
C
o.41 "i
0.3 ! "!
i
LM
0.2
0.1 ~iJ
o -
200 220 240 260 280 300 320 340 360

Wavelength (NM)
1.0
0.9 i
0.8
0.7
0.6
d
~ o.s
"~
c
0.4
W 0.3
0.2
~
200 220 240 260 280 300 320 340 360
Wavelength (NM)
Fig. 4. Ultraviolet spectrum of: A, Moiety M-2 in ethanol ( ) and in 0.003N NaOH in
ethanol ( .... ); B. Moiety M-2 in ethanol saturated with sodium acetate ( ) and in 0. IN
NaOH in ethanol ( .... ). Moiety M-2 was liberated by/3-glucosidase treatment of supernatant
obtained from tobacco cells incubated with 14 C~-naphthyl-labeled carbaryl. Spectra were
obtained as described in "Materials and methods."
of N-CH2OH-carbaryl conjugates, in view of the experimentally demonstrated con-

version of authentic N-CH2OH-carbaryl to desmethylcarbaryl by acid hydrolysis
(Figure 2).
The remainder of the organic extract of the acid hydrolysate was subjected to
1D-P-TLC. A moiety (M-3) was isolated which represented 0.5% of the recovered
radioactivity, or 0.021% of the added carbaryl (Table IV). M-3 had an R~ value of
0.45 with ID-P-TLC in System VIII. On the same TLC plate, trans-5,6-dihydro-
5,6-dihydroxy-l-naphthol had an Rf value of 0.73. All of the moieties listed in
Table I, as well as all of the naphthalenediols tested, had Rf values greater than that
of M-3 under the same conditions. Analysis of M-3 by high-resolution mass spec-
trometry (Shrader Analytical and Consulting Laboratories, Inc.) showed an apparent
molecular ion (M) at m/e = 178, with an elemental composition of C10H1003.
Fragment ions were observed at rn/e = 177 (M - H) and at rn/e = 161 (M - OH) with
elemental compositions of C~0H903 and C~0HgO~_, respectively. Collectively, these
data suggested that M-3 represented a cis-dihydrodiol of 1-naphthol which was
chromatographically different from the trans-5,6-dihydrodiol.
Moiety M-4, also isolated from this organic extract, represented 6% of the
recovered radioactivity or 0.240% of the added carbaryl (Table IV). High-resolution
mass spectrometry of M-4 indicated the presence of two apparent molecular ions of
elemental compositions CzrH44 and Cl0HsO, corresponding to 3,5-cholestadiene and
1-naphthol, respectively. These data could not reflect co-chromatography o f choles-
terol with 1-naphthol in System VIII, since cholesterol yields a m~lecular ion of
C27H460 with mass spectrometry (Stenhagen et al. 1969) which was not observed in
M-4; furthermore, authentic 1-naphthol had an Re value of 0.92 in System VIII,
while the Rf value for M-4 was 0.67. In addition, when 14Cl-naphthol was added to
SN from cells incubated with nonradioactive carbaryl and the mixture was acid
hydrolyzed, no M-4 could be detected in the organic extract of the hydrolysate.
These data would suggest O-l-naphthyl-cholesterol (cholest-5-en-3/3-yl-l-naphthol)
as a tentative structure for M-4. Moiety M-4 could not be extracted from non-
hydrolyzed SN. In addition, the proposed tentative structure for M-4 contains no
free hydroxyl groups. It is therefore possible that M-4 represents a dehydration
product of the actual metabolite. This metabolite could conceivably be a conjugate
of 1-(O-cholesteryl)-l,2-dihydro-2-hydroxynaphthalene or one of its isomers. The
thermal decomposition of this novel "detoxification" conjugate during mass spec-
trometry to yield 1-naphthol and cholestadiene followed the general pattern observed
with ammonium salts of steroid sulfates (Joseph et al. 1966) Moiety M-4 rep-
resented 3.02% of the total moieties isolated from tobacco cell cultures incubated
with 14Cl-naphthyl-labeled carbaryl (Table IV).
G. Moieties liberated by direct acid "hydrolysis of SN. Of the radioactivity

contained in the hydrolysate, 25.1% could be extracted into methylene chloride.
There was no loss of radioactivity due to hydrolysis. Since 10.9% of the radioactiv-
ity contained in non-hydrolyzed SN proved to be organoextractable, 14.2% of the
radioactivity contained in the SN represented conjugates labile to acid hydrolysis.
When an aliquot of the organic extract of the acid hydrolysate was subjected to
the TLC procedure for the detection of N-hydroxycarbaryl as its acetate derivative,
no radioactivity could be detected in the area corresponding to a standard of the
derivative. Radioactivity was detected in areas corresponding to standards of
1-naphthol and 1-naphthyl acetate; however, much of the radioactivity remained
near the origin, as did most of the standards subjected to this procedure (Table I).
The remainder of the organic extract was subjected to 1D-P-TLC, and radioac-
tivity co-chromatographing with standards of N-hydroxycarbaryl, 1-naphthol, or
carbaryl was further investigated: the radioactivity co-chromatographing with a
carbaryl standard was identified as authentic carbaryl by the mass (SR: Damico and
Benson 1965) and IR (SR: Chen and Benson 1966) spectra of the isolated product;
that co-chromatographing with a standard of N-hydroxycarbaryl was identified as
authentic desmethylcarbaryl by its IR (Figure 5-A) and mass (Figure 6) spectra.
2.5 3 4 5 6 7 8 9 10 12 15 20 30 40 50
lOO ~ ~J~ ~ ~1~
8o S
r h,,,Nl1" t
/,
60
( !yVw J
~-
-
20 ~/
V /
A
0
4000 3500 3000 2500 2000 1600 1600 1400 1200 1000 800 600 400 200
Wavenumber (CM- 1 )
2.5 3 4 5 6 7 8 9 10 12 15 20 304050
100 ~..--~ , i i i ~ , ,. ,|
80 ij ~
r
60 r~
', I / 'V '
E 40 O--C--NH 2
}--
P
20 @ , t
B
0 i I
4000 3500 3000 2500 2000 1800 1600 1400 1200 1000 800 600 400 200
Fig. 5. Infrared spectrum of: A. desmethylcarbaryl isolated from the acid hydrolysate of
supernatant obtained from tobacco cells incubated with t~Ct-naphthyl-labeled carbaryl; and
B. authentic desmethylcarbaryl. Spectra were obtained as described in "'Materials and
methods."
Radioactivity that co-chromatographed with authentic 1-naphthol was treated with

acetic anhydride. The l-naphthyl acetate thus formed was purified by repetitive TLC
and identified by a comparison of its IR (Figure 7-A) and mass (Figure 8) spectra
with those of an authentic standard.
A fluorescent material, originally thought to be radiolabeled and designated F - l ,

had a R~ value of 0.22 with TLC. Although the final extract was not radioactive, it
was analyzed by mass, UV, and IR spectrometry. All of these spectra were identical
to those of authentic 6-methoxy-7-hydroxycoumarin (scopoletin). The 7-O-glucoside
of this moiety is scopolin, a compound known to occur in tobacco (Yang et al.
1958) and in tobacco cell callus cultures (Fritig et al. 1966).
The area from one cm below to two cm above the origin was scraped and eluted
after the ID-P-TLC of the organic extract of the acid hydrolysate. The organic
extract of this area was subjected to 1D-P-TLC in System VIII, and 60% of the
recovered radioactivity had an Rf value of 0.30. This moiety was designated M-5
and was subjected to purification by TLC. Since insufficient material was recovered
for spectrometric analyses, M-5 was not further investigated.
M e t a b o l i s m of 14CO-labeled c a r b a r y l . A. Distribution o f radioactivity in cul-

ture fractions. After incubation of tobacco cell suspension cultures with 14CO-
labeled carbaryl, the added radioactivity was distributed among the various culture
fractions as shown in Table II. When tobacco ceils were homogenized, the radioac-
tivity was distributed among the various cell fractions as shown in Table III.
100- O
11 / H
O--C-N
75-
.~ 50 -
n"
25-
I I I 1
,t, ,l,,
I i I i i I 1
I
I [ I I ,
L
,
0 20 40 60 80 100 120 140 160 180 2 0

m/e ratio
Fig. 6. Mass spectrum of the desmethylcarbaryl isolated from the acid hydrolysate of
supernatant obtained from tobacco cells incubated with ~4Ct-naphthyl-labeled carbaryl. The
spectrum was obtained with an Atlas CH-4B mass spectrometer, as described in "Materials
and methods."
B. Moieties contained in acid-hydrolyzed tobacco cell supernatant (SN). No loss

of radioactivity from the hydrolysate was observed. After hydrolysis, 30.6% of the
radioactivity contained in the hydrolysate was organoextractable. Of this amount,
9.9% was shown by analytical TLC in two 2D-TLC systems to represent carbaryl.
Proof that this carbaryl was unconjugated will be presented in the '~Discussion"
section. Therefore, 20.7% of the radioactivity contained in the acid hydrolysate
represented moieties liberated from acid-labile conjugates contained in the SN.
When aliquots of the organic extract of the acid hydrolysate were subjected to
analytical TLC in two 1D-TLC systems, desmethylcarbaryl and carbaryl represented
30.5 and 32.5% of the recovered radioactivity, respectively. Thus, 0.153% of the
added carbaryl represented acid-labile conjugates of N-CH.,OH-carbaryl contained in
the SN and cell debris washes. When an aliquot of the organic extract was subjected
to the TLC procedure for the detection of N-hydroxycarbaryl as its acetate deriva-
tive, no radioactivity could be detected at the position corresponding to a standard
100 2.5 3 4 5 6 7 8 9 10 12 15 20 3 0 4 0 5 0
g 8o
c 60
g 40
~- 2O
4000
'"
A
3500
[
3000 2500
' I
2000 1800 1600 1400 1200 1000 800 600 400 200
2.5 3 4 5 6 7 8 9 10 12 15 20 3040 50
1 O0
80 --V ~ "-~- ----''~'~ c~ i

v
~ 60
o=
,, v ,L (,! 1
O--C--CH 3
~ 40
@ "
~ 20
B
0 , I I
4000 3500 3000 2500 2000 18(~0 1600 1400 1200 1000 800 6130 400 200
Fig. 7. Infrared spectrum of: A. the laC~-naphthyl-labeled l-naphthyl acetate formed from
l-naphthol liberated from tobacco cell supernatant by acid hydrolysis; and B. authentic
l-naphthyl acetate. Spectra were obtained as described in "Materials and methods."
of the derivative. All of the radioactivity remained near the origin, as did standards
of carbaryl and desmethylcarbaryl (Table [).
Authentic N-hydroxycarbaryl was added to the organic extract of the acid hyd-
rolysate, and radioactivity co-chromatographing with the added standard following
ID-P-TLC was isolated. When this isolated radioactivity was subjected to analytical
T L C in two 2 D - T L C s y s t e m s , 67% of the r e c o v e r e d r a d i o a c t i v i t y co-
chromatographed with a standard of desmethylcarbaryl in both cases. No radioactiv-
ity co-chromatographed with a standard of N-hydroxycarbaryl.
Following ID-P-TLC, radioactivity in the organic extract of the acid hydrolysate

to which authentic N-hydroxycarbaryl had been added co-chromatographed with a
standard of carbaryl. In two 2D-A-TLC systems, 88% of the recovered radioactivity
co-chromatographed with a standard of carbaryl. Thus, the only identifiable
moieties contained in the acid hydrolysate of SN obtained from cells incubated with
14CO-labeled carbaryl were desmethylcarbaryl and carbaryl.
Metabolism of N-14CH3-1abeled carbaryl. A. Distribution of radioactivity in

culture fractions. After incubation of suspension cultures o f tobacco cells with
N-14CH3-1abeled carbaryl, the radioactivity was distributed among the culture frac-
tions as shown in Table II. When the washed cells were homogenized, the cellular
radioactivity was distributed as shown in Table III.
B. Metabolites contained in the culture medium. When untreated culture medium

was extracted, 99.3% of the radioactivity was recovered in the organic phase. An
0
1 O0 - II
O-C-CH 3
75-
c
5o-
"3
E
25-
,i ~I I II LI
i I i | I ! I I I I I 1 I I I
0 20 40 60 80 100 12o 140 160 180 200
m/e ratio
Fig. 8. Mass spectrum of the l-naphthyl acetate formed from laC~-naphthyl-labeled

l-naphthol liberated from tobacco cell supernatant by acid hydrolysis. The spectrum was
obtained with an Atlas CH-4B mass spectrometer, as described in "Materials and methods."
aliquot of the organic extract was subjected to 2D-A-TLC and 96.2% of the reco-
vered radioactivity co-chromatographed with a standard of carbaryl. Of the remain-
ing radioactivity, 2.6% co-chromatographed with N-CH2OH-carbaryl and 0.1% with
7-hydroxycarbaryl standards. Thus, 2.230% of the added carbaryl had been con-
verted to N-CH2OH-carbaryl by the cells, and was presumably excreted unconjugated
into the culture medium (Table VI). Similarly, unconjugated 7-hydroxycarbaryl
contained in the culture medium and cell washes represented 0.086% of the added
carbaryl.
The remainder of the organic extract of the culture medium was subjected to
1D-P-TLC, and radioactivity co-chromatographing with N-CH2OH-carbaryl was iso-
lated. This material was further purified by TLC and identified as authentic
N-CH,.OH-carbaryl by IR spectrometry (Figure 3-C).
C. Metabolites contained in untreated SN. The entire SN was extracted before

acid hydrolysis, and 0.4% of the radioactivity appeared in the organic phase.
Table V. Organic moieties recovered from the acid hydrolysate of cell supernatant
(and cell debris washes) obtained from tobacco cells incubated with
14Cl-naphthyl-labeled carbaryl a
% of
% of added total characterized
-~Ioiety b radioactivity moieties
l-Naphthol 1.72 l 61.35

Desmethylcarbaryle 0.863 30.77
M-5 (Unknown) 0.22 l 7.88
Total characterized metabolite
moieties 2.805 100.00
Carbaryl (unconjugated) a t.551
Unknown water-solubles e 14.152
Unknown organoextractables 0.386
(by difference)
Total radioactivity recovered
from cell supernatant and
cell debris washes 18.894
aThe separation of cell culture fractions is described in "Materials and methods;" metabolites
contained in the cells were investigated utilizing acid hydrolysis, as described in Section B.
methods."
eTaken to represent the moiety resulting from acid hydrolysis of conjugates of N-CHzOH-
carbaryl, as discussed in "Results."
aSee "Discussion" section and Table IV for proof of nonconjugation.
~Radioactivity remaining in the water phase following extraction of the acid hydrolysate.
Aliquots o f the organic extract were subjected to analytical TLC in two 2 D - T L C

systems. Of the recovered r a d i o a c t i v i t y , 75% c o - c h r o m a t o g r a p h e d with a standard
o f carbaryl in both systems, while 0.5% c o - c h r o m a t o g r a p h e d with a standard o f
N-CH.,OH-carbaryl. Thus, 0 . 0 0 0 3 % o f the a d d e d carbaryl was present as unconju-
gated N - C H 2 O H - c a r b a r y l in the S N and cell debris washes (not entered in T a b l e VI).
T a b l e VI. Organic moieties recovered from the tobacco cell cultures incubated with
N-14CH3-labeled carbaryl a
% of total
% of added characterized
Moiety b Form radioactivity moieties
N-CH2OH-Carbaryl Unconjugated e'a 2.230 95.75

7-OH-Carbaryl Unconjugated c'a 0.086 3.70
Total
conjugate(s) c'f 0.001 0.04
4-OH-Carbaryl Total
conjugate(s) e'r 0.008 0.34
5-OH-Carbaryl Total
conjugate(s) e.f 0.004 0.17
Total characterized metabolite moieties 2.329 100.00
Carbaryl Unconjugated c'a 85.535
Unconjugated ~.g 0.052
Unknowns Water-solublesh 12.090
Water-solublesi 0.605
Cell debris 1.400
Organoextractables
(by difference 0.318
Total recovered radioactivity I00.000
aThe separation of cell culture fractions is described in "Materials and methods;" metabo-
lites contained in the cells were investigated utilizing acid hydrolysis, as described in
Section D.
methods."
eCould be extracted without hydrolytic treatments.
aObtained from the organic extract of untreated culture medium and cell washes.
eObtained by direct acid hydrolysis, with no prior treatment with /3-glucosidase.
fObtained from the organic extract of the acid hydrolysate of cell supernatant and cell debris
washes.
gSee the "Discussion" section and Table IV for proof of nonconjugation.
hRadioactivity remaining in the water phase following extraction of the acid hydrolysate of
cell supernatant and cell debris washes.
iRadioactivity remaining in the water phase following extraction of untreated culture medium
and cell washes.
D. Moieties liberated by acid hydrolysis of SN. The water phase from extraction
of the SN was acid hydrolyzed, and the hydrolysate was extracted. Only 0.9% of the
recovered radioactivity was organoextractable, and there was no loss of radioactivity
due to hydrolysis. When a standard of N-hydroxycarbaryl was added to an aliquot of
the organic extract of the acid hydrolysate, and the mixture was subjected to
1D-A-TLC, no radioactivity appeared in the UV-absorbing spot corresponding to the
added standard.
Standards of N-hydroxycarbaryl, carbaryl, and 4-hydroxycarbaryl were added to

the remainder of the organic extract, and the mixture was subjected to 1D-P-TLC.
No radioactivity was contained in the extract of the material corresponding to the
added N-hydroxycarbaryl. Radioactivity contained in the remaining eluates was then
characterized by 2D-A-TLC. Based on the total radioactivity recovered from the
original preparative TLC plate, the following amounts of radioactive moieties were
identified in the extract of the acid hydrolysate of the SN: carbaryl (47%);
4-hydroxycarbaryl (7%); 5-hydroxycarbaryl (4%); 7-hydroxycarbaryl (1%); and un-
knowns (41%). The percentages of the added carbaryl which acid-labile conjugates
of these moieties (contained in the SN and cell debris washes) represented are given
in Table VI.
Discussion
Metabolism of carbaryl by tobacco cells in suspension culture. The proposed
scheme for the metabolism of carbaryl by tobacco cells in culture is presented in
Figure 9. Hydrolysis of the carbamate moiety of carbaryl and conjugation of the
resulting l-naphthol represented a major pathway of carbaryl metabolism in these
cells. From data obtained with the use of 14C~-naphthyl-labeled carbaryl, 72.16% of
the total characterized metabolites represented conjugates of 1-naphthol contained in
the SN and cell debris washes (Table IV). When tobacco cells were incubated with
~4CO-labeled carbaryl, 47.3% of the added radioactivity was presumably lost as
14CO2 (Table II). This observation suggested extensive hydrolysis and decarboxyla-
tion of the carbamate ester and was in good agreement with the observed production
of conjugates of 1-naphthol from ~4Cl-naphthyl-labeled carbaryl. No unconjugated
14C,-naphthyl-labeled 1-naphthol could be detected in any culture fraction, indicat-
ing that conjugation mechanisms were not rate-limiting factors in this metabolic
pathway.
Approximately 46% of the 14Cl-naphthyl-labeled 1-naphthol conjugates contained

in the SN were labile to /3-glucosidase treatment, indicating probable conjugation
with glucose or other sugars (Table IV). A combination of aryl sulfatase and
/3-glucosidase was only very slightly, if at all, more effective than fl-glucosidase
alone in the hydrolysis of metabolite conjugates contained in the SN. Similar
behavior has been observed with metabolite conjugates produced from carbaryl by
intact bean plants (Kuhr and Casida 1967). The remaining 54% of the isolated
1-naphthol conjugates were not labile to/3-glucosidase, but were labile to 0.1N HCI
hydrolysis (Table IV). The nature of the conjugation system(s) producing these
90 R. K. Locke et al.
jCH3
O-C-N
@ .
(100%)
Carbaryl
(Cells end culture medium) O
II J CH2OH
O-C--N
[1,co21j
(Air)
(18%)
(Culture medium)
(Cell supernatant)
O
II J CH20-CONJ
O-CONJ O--C--N
(72%) (2.5%)
O O
II ~ CH3 II 7 cH3
O--C--N O--C--N
C O N ~ ~H
10.8%1e (0.3%) e
O-CONJ
CH3
H3
0
II ~ CH3
O--C--N O-CONJ CH3
(0.3%~
O-CONJ H
H OH
Fig. 9. Tentative scheme of carbaryl metabolism by tobacco cells in culture. Numbers in

parentheses refer to percentages based on the total 14Ct-naphthyl-labeled moieties charac-
terized (see Table IV). An asterisk (*) indicates that the structure of the moiety has been
characterized by infrared spectrometry; (A) by mass spectrometry; (o) by analytical thin-layer
chromatography; and CON J- indicates conjugation. The positions of the hydroxyl groups and
conjugation position(s) are unknown in the moiety characterized as a dihydrodiol of naphthol
(M-3; Table IV); however, the moiety is not the trans-5,6-dihydrodiol of l-naphthol. As
discussed in '+Results," the moiety characterized as the l-naphthyl ether of cholesterol (M-4;
Table IV) is thought to be liberated by acid hydrolysis from a conjugate of l-(O-
cholesteryl)-1,2-dihydro-2-hydroxynaphthalene or a similar dihydrodiol conjugate.
acid-labile metabolites is unknown. Amino acid conjugates would be likely, how-

ever, since this mode of conjugation is well known in plants (Andreae and Good
1957; Bach 1961; Bach and Fellig 1961, Kaslander et al. 1962, Kl~imbt 1961,
Massini 1959, Feung et al. 1973). It must be recognized that the 1-naphthol moiety
could arise not only from conjugates of 1-naphthol but also from conjugates of
naphthalene dihydrodiols.
The second most abundant moiety produced by tobacco cells incubated with
14C1-naphthyl-labeled carbaryl was N-CH2OH-carbaryl, which was present in
21.04% of the characterized metabolites (Table IV). Of the total N-CHzOH-carbaryl
moiety isolated, 87.39% was found in the unconjugated form in the culture medium
and cell washes. An additional 0.70% was found in the unconjugated form in the SN
and cell debris washes, fl-Glucosidase-labile conjugates of N-CH2OH-carbaryl con-
tained in the SN and cell debris washes represented an additional I1.44%, while
conjugates not labile to fl-glucosidase but labile to acid represented 0.47% of the
total N-CHzOH-carbaryl moiety isolated.
The fact that the major portion of the 14Cl-naphthyl-labeled N-CHzOH-carbaryl

moiety was found in the unconjugated form in the culture medium may indicate that
conjugation mechanisms were the rate-limiting factors in this metabolic pathway.
Furthermore,/3-glucosidase-labile conjugates of N-CH2OH-carbaryl were predomin-
ant, unlike the predominance of non-/3-glucosidase-labile conjugates in the case of
the 1-naphthol moiety (Table IV). The radioactivity found as unconjugated
N-CH2OH-carbaryl in the culture medium and cell washes represented 1.459% of
the added carbaryl with the use of the 14C~-naphthyl-labeled compound (Table IV).
This was in good agreement with the figure of 2.230% obtained with the use of
N-X4CHa-labeled carbaryl for the unconjugated N-CH2OH-carbaryl contained in the
same culture fraction (Table VI).
With direct acid hydrolysis of SN obtained from cells incubated with 14C~-
naphthyl-labeled carbaryl, the second most abundant characterized moiety was
desmethylcarbaryl (Table V). Desmethylcarbaryl was the only moiety, excluding
carbaryl, which was isolated from the acid hydrolysate of SN from tobacco cells
incubated with 14CO-labeled carbaryl. The desmethylcarbaryl moiety probably re-
sulted from hydrolysis of conjugates of N-CHzOH-carbaryl, as previously discussed
(Figure 2).
The third most abundant moiety liberated by direct acid hydrolysis of SN ob-
tained from cells incubated with 14Cl-naphthyl-labeled carbaryl was M-5, which
represented 7.88% of the total moieties liberated (Table V). Since insufficient
material was recovered following preparative TLC to permit spectrometric analyses,
M-5 has been characterized by TLC Rf values only. Its TLC behavior indicated that
M-5 was a very polar molecule and was different from trans-5,6-dihydro-5,6-
dihydroxy- 1-naphthyl methylcarbamate and trans-5,6-dihydro-5,6-dihydroxy- l-
naphthol.
Ring hydroxylation of carbaryl and subsequent conjugation of the hydroxylated

methylcarbamates represented a relatively minor pathway of carbaryl metabolism in
tobacco cells. ~4C1-Naphthyl-labeled conjugates of 7-hydroxycarbaryl represented
0.82% of the total characterized moieties from all culture fractions, whileconjugates
of 4-hydroxy- and 5-hydroxycarbaryl each represented 0.30% (Table IV). These
hydroxycarbaryl moieties were also detected in the acid hydrolysate of the SN
obtained from ceils incubated with N-~4CH3-1abeled carbaryl (Table VI). In both
cases, these moieties were identified only by 2D-A-TLC.
Moiety M-I was obtained by organic extraction of SN obtained from tobacco

cells incubated with 14C~-naphthyl-labeled carbaryl. Following purification by re-
petitive preparative TLC, the ethyl acetate extract was analyzed by Fourier-
transform NMR, IR, and mass spectrometry. The identity of M-1 is as yet unknown;
however, the preliminary data would be consistent with a hydroxylated 1,4-
dihydro-1,4-epiperoxynaphthalene. Studies are currently in progress to elucidate the
structure of M-I, which represented 1.36% of the characterized moieties (Table IV).
Moiety M-2 was liberated by /3-glucosidase treatment of SN obtained from cells
incubated with ~4C~-naphthyl-labeled carbaryl, and represented 0.74% of the total
isolated moieties from all culture fractions (Table IV). Insufficient material was
recovered after TLC purification to permit the analysis of M-2 by mass or IR
spectrometry; however, the UV spectrum of M-2 was different from all of the
standards tested and suggested that the molecule contained an ionizable group(s) and
an aromatic ring structure (Figure 4). With 1D-TLC in System VIII, both M-2 and
M-5 had an Rt value of 0.30, while both remained near the origin with 1D-TLC in
System XIV. These data suggest that moieties M-2 (/3-glucosidase-labile) and M-5
(acid-labile) may represent the same compound.
Moiety M-3 was tentatively identified by high-resolution mass spectrometry and

TLC characteristics as a cis-dihydrodiol of 1-naphthol. This moiety was liberated by
acid hydrolysis of the water phase following extraction of the /3-glucosidase hyd-
rolysate of SN obtained from cells incubated with 14C1-naphthyl-labeled carbaryl.
Moiety M-3 represented 0.26% of the total characterized moieties from all culture
fractions (Table IV), and was shown not be trans-5,6-dihydro-5,6-dihydroxy-1-
naphthol by 1D-A-TLC.
Conjugation with cholesterol as a possible "detoxification m e c h a n i s m " in

tobacco cells. Although conjugates of cholesterol and endogenous natural products,
such as sulfate and glucuronic acid, have been known for some time in mammalian
systems (Bernstein and Solomon 1970), to our knowledge the conjugation of
exogenous compounds (xenobiotics) with sterols has never been demonstrated as a
"detoxification mechanism" in any biological system. On the basis of high-
resolution mass spectrometry we have tentatively identified moiety M-4, represent-
ing 3.02% of the total characterized moieties from tobacco cell cultures incubated
with 14Cl-naphthyl-labeled carbaryl (Table IV), as the 1-naphthyl ether of choles-
terol (cholest-5-en-3/3-yl-l-naphthol). This tentative assignment of structure must,
of course, be verified by other physical methods. As discussed in "Results," moiety
M-4 is thought to represent a dehydration product of the actual metabolite. This

metabolite may be a sugar or amino acid conjugate of 1-(O-cholesteryl)-l,2-
dihydro-2-hydroxynaphthalene or one of its isomers. Cholesterol is known to occur
in tobacco plant leaves (Stedman 1968) and has been identified by mass spec-
trometry in callus cultures of tobacco cells (Benveniste et al. 1966).
Ethereal cholesterol plant conjugates might pose a toxicological hazard for man
with regard to possible interference with enzyme systems involved in mammalian
utilization and deposition of cholesterol. Excellent reviews are currently available
on the utilization of cholesterol and the biological effects of the known conjugated
steroids (Bernstein and Solomon 1970, Layne 1970, Mason 1970, Pasqualini 1970,
Scherst~n 1970). However, little is known about the possible biological effects of
uncharged ethereal "detoxification conjugates" of steroids.
The non-production of N-conjugates of carbaryl by tobacco cells. Significant

amounts of carbaryl could be extracted from enzymic or acid hydrolysates of SN
obtained from tobacco cells incubated with carbaryl. Similar results have been
reported by other workers in studies involving the metabolism of carbaryl by several
species of intact plants (Andrawes et al. 1972, Kuhr and Casida 1967, Dorough and
Wiggins 1969, Wiggins et al. 1970). The occurrence of the carbaryl moiety in
hydrolysates has been postulated to result from the hydrolysis of N-conjugates of
carbaryl (Kuhr and Casida 1967) or of carbaryl complexes with natural plant
pigments (Mumma et al. 1971).
In our studies with ~4C~-naphthyl-labeled carbaryl, the total amount of carbaryl

recovered from the fl-glucosidase hydrolysate of tobacco cell SN, and from the acid
hydrolysate of the water phase obtained from extraction of the enzymic hydrolysate,
was slightly less than the amount of carbaryl which could be obtained by extraction
of untreated SN (Table IV).. Thus, all the carbaryl obtained from enzymic or acid
hydrolysates of the SN could be ascribed to unconjugated carbaryl contained in the
cells prior to biotransformation.
The non-production of N-O-conjugates of N-hydroxycarbaryl from carbaryl

by tobacco cells. The N-hydroxycarbaryl moiety was not detected in any tobacco
cell culture fraction. After incubation of tobacco cells with each of the three
radiolabeled carbaryls, no radioactivity was found to co-chromatograph with an
authentic standard of N-hydroxycarbaryl in the 2D-A-TLC of organic extracts of any
culture fraction. Furthermore, when these organic extracts were subjected to the
TLC procedure for the detection of N-hydroxycarbaryl as its acetate derivative, no
radioactivity co-chromatographed with the derivative formed from authentic
N-hydroxyc'arbaryl.
In the case of cells incubated with 14Cl-naphthyl- or 14CO-labeled carbaryl, the

radioactivity contained in organic extracts of acid hydrolysates of SN which co-
chromatographed with authentic N-hydroxycarbaryl following 1D-A-TLC in Sys-
tems XIII or XIV was shown to be desmethylcarbaryl. No radioactivity co-
chromatographing with authentic N-hydroxycarbaryl could be detected in the same

fractions and under the same conditions with cells incubated with N-14CI-I3-1abeled
carbaryl.
Desmethylcarbaryl was previously reported to co-chromatograph with

N-hydroxycarbaryl on 250-/z commercial TLC plates in System XIII, but pure
standards of these two compounds were separated by 1.5 cm in System XIV under
the conditions described (Locke 1972b). Apparently organoextractable natural pro-
ducts present in acid hydrolysates of the SN decreased the ID-TLC separation of
N-hydroxycarbaryl and desmethylcarbaryl observed in System XIV with pure stan-
dards. However, this difficulty was overcome by the use of 2D-A-TLC (System XIV
followed by System XIII).
Thus the moiety tentatively identified as N-hydroxycarbaryl in preliminary

studies of the metabolism of carbaryl by tobacco cells in culture (Locke 1972a) was,
in fact, desmethylcarbaryl. The desmethylcarbaryl probably resulted from the acid
hydrolysis of conjugates of N-CH~OH-carbaryl.
Comparison of the metabolites produced from carbaryl by the tobacco cell in

vitro system with those produced by plants in vivo. Since data about the in vivo
metabolism of carbaryl by Nicotiana tabacum L. ear. Xanthi are currently unavaila-
ble, no direct qualitative or quantitative comparisons can be made with regard to the
metabolites produced in vitro by the XD line of tobacco cells and those produced in
vivo by the plant species from which the line was derived. Nonetheless, the in vitro
tobacco cell system qualitatively reflected the in vivo metabolism of carbaryl by
several of the higher plant species thus far investigated.
Except for conjugates of l-naphthol, conjugates of N-CH~OH-carbaryl rep-

resented the major metabolites (21.04%) produced from carbaryl by tobacco cells.
Such conjugates have been characterized as major metabolites in studies of the
metabolism of carbaryl by intact corn, tomato, potato, alfalfa, peanut, and wheat
plants (Andrawes et al. 1972), as well as by bean plants (Kuhr and Casida 1967,
Mumma et al. 1971, Wiggins et al. 1970) and by pea plants (Mumma et al. 1971).
The major portion of the N-CH2OH-carbaryl moiety produced by tobacco cells was
in the unconjugated form (Table IV), suggesting a deficiency in enzymic conjuga-
tion systems with respect to this moiety. A similar deficiency may exist in intact
cotton plants (Mostafa et al. 1966).
Conjugates of l-naphthol represented 72.16% of the total characterized metabo-

lites produced from carbaryl by tobacco cells. Mostafa et al. (1966) showed that
hydrolysis of the carbamate group represented a major (47%) pathway of carbaryl
metabolism in intact cotton plants; 25% of the total metabolites was identified as
unconjugated 1-naphthol. In addition, several investigators have identified signific-
ant amounts of the 1-naphthol moiety with the metabolism of carbaryl by several
species of whole plants (Andrawes et al. 1972, Dorough and Wiggins 1969, Wig-
gins et al. 1970).
Conjugates of 7-, 5-, and 4-hydroxycarbaryl collectively represented only 1.42%

of the total characterized metabolites produced from carbaryl by tobacco cells (Table
IV). Such conjugates have been shown to represent a greater percentage of the total
metabolites in several species of intact plants (Andrawes et al. 1972, Mumma et al.
1971, Wiggins et al. 1970).
Several investigators have characterized the trans-5,6-dihydro-5,6-dihydroxy-1-

naphthyl methylcarbamate moiety as a minor component of conjugates obtained in
whole plant studies (Andrawes et al. 1972, Kuhr and Casida 1967, Wiggins et al.
1970) or in studies with plant cell cultures (Locke et al. 1971). In the present study~
we have shown by 2D-A-TLC that /3-glucosidase-labile conjugates of trans-5,6-
dihydro-5,6-dihydroxy-naphthyl methylcarbamate and trans-5,6-dihydro-5,6-
dihydroxy-l-naphthol present in tobacco cell SN represented less than 0.03 and
0.01% of the radioactivity added as 14C~-naphthyl-labeled carbaryl, respectively.
However, by mass spectrometry we have tentatively identified moiety M-3, which
represented 0.26% of the total metabolites produced from 14C~-naphthyl-labeled
carbaryl by tobacco cells (Table IV), as a cis-dihydrodiol of 1-naphthol other than
the trans-5,6-dihydrodiol.
Usefulness of the in vitro tobacco cell system for studies of pesticide

metabolism by higher plants. Although the actual toxicological hazard posed for
mart by pesticide residues found in higher plants must always be assessed under field
conditions in order to determine the nature of the residue complex under conditions
of natural weathering, penetration, photodecomposition, and translocation, the use
of in vitro plant cell systems for determining the nature of the metabolites produced
from pesticides by higher plants has very definite advantages. The use of suspen-
sion cultures of plant cells in pesticide metabolism studies obviates the difficulties
of poor penetration and translocation frequently encountered in in vivo plant studies
and results in a much greater amount of metabolite production. Frequently a suffi-
cient amount of radiolabeled metabolites can be obtained from one suspension
culture to allow conclusive structural determinations by IR and mass spectrometry in
conjunction with derivatization and chromatographic techniques.
In vitro plant cell systems also provide a means for determining the metabolites
produced from pesticides by higher plants in the absence of concomitant metabolism
by soil microorganisms. If the plant cell line is propagated in the dark, the effects of
photochemical reactions are also circumvented. In the case of herbicides, the use of
a plant cell line whose biochemistry has been extensively studied permits meaning-
ful insights into mechanisms of action.
The XD cell line of tobacco cells, derived from Nicotiana tabacum var. Xanthi
(Filner 1965), possesses certain advantages over other plant cell lines with regard to
pesticide metabolism studies. The tobacco cell line proliferates well in a completely
defined and inexpensive medium (M-1D; Filner 1965) which includes the auxin
2,4-dichlorophenoxyacetic acid, salts, vitamins, and sucrose. Tobacco ceils also
grow well in the dark, thus eliminating photochemical effects upon compounds
whose metabolism is under investigation.
Since its isolation in 1961 (Filner 1965), the XD line of tobacco cells (also
referred to as the WR-132 cell line by certain authors) has been extensively investi-
gated with regard to many biochemical parameters (De Jong et al. 1967a, 1967b,
1968, Filner 1965, 1966, Hart and Filner 1969, Olson 1964, Trosko and Mansour
1968). This makes its use in studies concerning the mode of action of herbicidal
compounds more attractive as compared with other cell lines whose biochemistry
has been less thoroughly investigated. As we do not contemplate further studies in
the area of pesticide metabolism, it is hoped that other investigators might become
interested in the use of the tobacco cell in v i t r o system for this purpose. The
metabolites produced from carbaryl by tobacco cells have adquately reflected the
metabolites reported for higher plants in in v i v o studies. Such was also the case with
regard to the metabolism of the only other pesticide which we have studied in the
tobacco cell system, 4-nitrophenyl ot,~,ot-trifluoro-2-nitro-p-tolyl ether (Locke and
Baron 1972).
Acknowledgments
The authors express appreciation to Dr. W. R. Benson, Mr. J. R. Evans, and

Miss J. E. Rothlein, Food and Drug Administration, for the synthesis of a portion of
the naphthyl-radiotabeled carbaryl used in this study, and microbiological and
chromatographic assistance, respectively. We are also grateful to Drs. W. J.
Bartley, J. A. Durden, and F. A. Richey, Jr., Union Carbide Corporation, for the
generous gift of many chromatographic standards and the radiolabeled carbaryls.
The authors also thank Drs. H. J. C. Yeh and D. M. Jerina, National Institutes of
Health, for obtaining the Fourier-transform nuclear magnetic resonance spectrum.
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Manuscript received March 28, 1974; accepted December 9, 1974.

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IDENTIFICATION BY PHYSICAL MEANS OF

ORGANIC MOIETIES OF CONJUGATES PRODUCED

Carbaryl (1-naphthyl methylcarbamate), labeled with ~4C in the C~-naphthyl, car-

Earlier reports from this laboratory ( L o c k e et al. 1971, L o c k e 1972a) have

Archives of Environmental Contamination 60

labeled carbaryl (l-naphthyl methylcarbamate) by tobacco cells in suspension cul-

Since carbaryl is a widely used insecticide, it was important to investigate by

Materials and methods

high-protein samples, except that the concentration of thixotropic gel (Cab-O-Sil)

Extraction procedure. To one volume of a given culture fraction, one volume of

The extraction of enzymic or acid hydrolysates of SN by this procedure fre-

Thin-layer chromatography. A. S o l v e n t systems and standards. Noncommercial

B. One-dimensional analytical TLC ( 1 D - A - T L C ) . For 1D-A-TLC, organic ex-

chromatographed simultaneously with unknowns in all cases. Compounds were

C. One-dimensional preparative TLC (1D-P-TLC). For the preparative isolation

D. Two-dimensional analytical TLC (2D-A-TLC). For 2D-A-TLC, organic ex-

E. Two-dimensional preparative TLC (2D-P-TLC). Certain organic extracts were

Thin-layer chromatographic procedure for the detection and separation of

to be acetylated were applied. The plate was subjected to 1 D - A - T L C in S y s t e m

Table I. Thin-Layer chromatographic procedure for the detection of

aprocedure described in "Materials and methods."

Infrared (IR) spectrometry. All IR spectra, except those of metabolite-derived

Mass spectrometry. Low-resolution mass spectra were generally obtained with

Ultraviolet (UV) spectrometry. UV spectra were obtained with a Beckman Acta

Radiolabeled compounds and culture conditions used. In all metabolism

A 30-ml portion of a ten-day suspension culture of tobacco cells and 100 ml of

To insure proper growth, suspension cultures of XD tobacco cells should initially

Two series of experiments were conducted with 14C~-naphthyl-labeled carbaryl,

Fractionation of suspension cultures. Following incubation, the cells were

Fractionation of radioactivity incorporated into tobacco cells. The tobacco

Investigation of metabolites contained in the culture medium. Filtered culture

The remainder of the organic extract of the medium containing N-~4CHa-labeled

Investigation of metabolites incorporated into tobacco cells. A. fl-Glucosidase

To establish the lability of radiolabeled conjugates contained in the SN to en-

A control was prepared by incubating an aliquot of SN under identical conditions,

The remainder of the SN was treated with/3-glucosidase only. To 346 ml of SN,

An aliquot of the organic phase from the extraction of SN incubated with

The water phase from the extraction of the/3-glucosidase hydrolysate of SN was

B. Acid hydrolysis of ~4C1-naphthyl-labeled cell metabolites. In the second series

An aliquot of non-hydrolyzed SN was extracted. The remainder of the SN was

Material co-chromatographing with a carbaryl standard (Rf = 0.42) was further

C. Acid Hydrolysis of 14CO-labeled cell metabolites. The SN obtained from

To the remainder of the organic phase, 160 /zg of authentic nonradioactive

It was noted that radioactivity in the organic extract to which authentic

. D. Acid hydrolysis of N-14CHa-labeled cell metabolites. The entire SN obtained

The acetone extracts of materials co-chromatographing with the added standards

Demonstration of the conversion of authentic N-CH2OH-carbaryl to

x4CI-Naphthyl-labeled N-CHzOH-carbaryl, previously obtained by/3-glucosidase

SN was freshly prepared by homogenizing untreated cells in water (1:1 w/v) as

Thin-layer chromatographic detection of N-hydroxycarbaryl. A. Formation

quantitatively converted by treatment with acetic anydride to a single product,

l-Naphthol was converted in good yield by the derivatization procedure to a

These data indicated that a sample suspected of containing the radiolabeled

B. 2D-TLC ofunderivatized N-hydroxycarbaryl. The two solvent systems used by

A 2D-TLC system developed by Wiggins et al. (1970) provided a good separa-

A modification of the 2D-TLC system developed by Wiggins et al. (1970),

Demonstration of the conversion of authentic N-CH2OH-carbaryl to des-

Metabolism of 14Cl-naphthyl-labeled carbaryl. A. Distribution of radioactivity

Fig. 2. Acid hydrolysis of authentic 14Ct-naphthyl-labeled N-CH.,OH-carbaryl in the presence

T a b l e II. Distribution of radioactivity in tobacco cell suspension cultures incubated

C1-Naphthyl 30.2 14.5 55.3 0.0 6.0 x 10-6

aCulture conditions are described in "Materials and methods."

C. Metabolites contained in the cell debris. Only 1% of the radioactivity con-