[CANCER RESEARCH 46, 3249-3253, July 1986]

Measurement of Aflatoxin Bi, Its Metabolites, and DNA Adducts by Synchronous

Fluorescence Spectrophotometry
Curtis C. Harris,1 Gerald LaVeck, John Groopman, Vincent L. Wilson, and Dean Mann
Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20S92 [C. C. H., G. L., K L. W., D. M.], and Boston
University School of Public Health, Department of Environmental Health, Boston, Massachusetts 02118 [J. G.J

ABSTRACT
Sensitive and specific methods are needed for measuring human ex
posure to carcinogens. Synchronous fluorescence Spectrophotometry can
be used to measure fmol of aflatoxins, their metabolites, and DNA
adducts. Computer-assisted analysis of spectra of these agents obtained
by synchronous fluorescence Spectrophotometry can be displayed as
contour maps which are highly specific for each agent. Individual agents
in mixtures, e.g. aflatoxins B, and Mcan be identified by fourth
derivative spectral analysis. This physical method should complement
immunological and other methods to measure aflatoxin BI, its metabo
lites, and nucleic acid adducts.

INTRODUCTION
Biochemical and molecular epidemiology is a multidiscipli
nar) area that combines laboratory and epidemiolgica! ap
proaches of cancer research (1, 2). The primary goal is to
identify individuals who are at high cancer risk by obtaining
evidence of (a) pathobiological lesions in target cells caused
by exposure to carcinogens and/or (b) increased susceptibility
to carcinogens due to either inherited or acquired host factors.
Carcinogen-DNA adducts are examples of pathobiological le
sions considered to be important in the early stage, i.e., tumor
initiation, and perhaps in a later stage, i.e., tumor conversion,
of carcinogenesis. Recently, both immunological and nonradiolabeling physical methods have been developed to measure
adducts in macromolecules including DNA isolated from car
cinogen-exposed tissues and cells (see review in Ref. 3). One of
the advantages of these approaches is that they can be specific
for both carcinogen and target tissue. Arguing that data ob
tained by 2 different analytical methods would be confirmatory
and more convincing than measurements from a single tech
nique, we describe here a refined physical method, SFS2 (4-7),
to compliment enzyme radioimmunoassays (8-11) for the mea
surement of aflatoxin B,, its metabolites, and DNA adducts.

product was dried in a vaccum. Preparation of AFBt-N7-mGua was by

the method described by Essigmann et al. (16) using dimethyl sulfate.
In Vivo Modified DNA. Male Fisher rats (ISO g) were given i.p.
injections of SO u\ of a mixture of '11-AI-'B, and unlabeled A1-"B,at
concentrations of 900, 138, or 62 fig/kg at a specific activity of 1.2 Ci/
mmol in dimethyl sulfoxide. After 2 h, the rats were killed by CO.,
narcosis, and the livers were removed and frozen at 70"C.
Isolation of DNA. DNA was isolated from rat liver by a modification
of the method of Murmur (13). Tissues were thawed and minced in
0.2x SSC buffer. After centrifugation at 1000 x g, the supernatant was
decanted and the tissue was washed in 0.2x SSC and recentrifuged
until the supernatant was clear. The tissue was then suspended in 5 ml
of 0.2x SSC and the cells were separated in a Dounce homogenizer.
After 3 washes in 0.2x SSC, the cell pellet was resuspended in 1 ml of
IX SSC, and 8 ml of 10 HIMTris (pH 7)-1 IHM EDTA-1 % sodium
dodecyl sulfate, were added to the pellet along with 10,000 units of
Pronase in 1 ml. After l h incubation at 45 C. 10,000 additional units
of Pronase were added and incubation was continued for 1 h. The
digested tissue was phenol extracted 3 times and once with chloroform/
isoamyl alcohol (24/1, v/v). After precipitation in ethanol and 1 i( I.
overnight at 4V. the DNA was carefully spooled on a glass rod and
washed 3 times in ethanol. The remaining ethanol was evaporated and
the pellet was resuspended in 10 mM I ris 1 DIMEDTA. The adduction
level of aflatoxin metabolites to DNA was determined by measurement
of tritium bound to the purified DNA and, following hydrolysis, HPLC
analysis.
Hydrolysis of DNA. Nucleic acids were adjusted to 0.15 N HC1 and
treated for 15 min at 90-95*C as detailed by Lin et al. (14). This

procedure releases >90% of the covalently bound radioactivity from

the modified DNA. Hydrolysates were rapidly cooled on ice, neutralized
with 1 M ammonium formate (pH S.O) and l N KOH, made 5%
methanol, and applied to dg-Sep-Pak (Waters Associated, Milford,
MA). The Sep-Pak was washed with 5% methanol to remove unhydrolyzed DNA and other polar compounds and then eluted with 80%
methanol to release the more lipophilic aflatoxin derivatives. The
methanol was removed from these samples by rotary evaporation under
reduced pressure and the resulting mixture was adjusted to 10% ethanol
prior to HPLC.
Chromatography. Nucleic acid hydrolysates were analyzed by HPLC
using an Ultrasphere-ODS Cm 5-pm column (Beckman Instrument
Co., Berkeley, CA) and a Beckman Model 322 liquid Chromatograph
MATERIALS AND METHODS
equipped with Model 160 detector (365 nm). Gradient chromatography
Chemicals. Aflatoxins B,, B2, Bj., M,, M2, GI, G2, 62,, PI, and Qi
was performed at ambient temperature with an elution buffer of 10%
and aflatoxicol were obtained from Sigma Chemical Company, St.
redistilled ethanol 20 mM triethylammonium formate (pH 3.0) to 18%
Louis, MO. Stock solutions were dissolved in chloroform and methanol
ethanol over 25 min at 1.0 ml/min. These procedures have been
and then diluted in 10 nisi Tris-I mMEDTA to working concentrations.
described previously (IS). One-min fractions were collected and tritium
3H-Aflatoxin BI (15 Ci/mmol) was obtained from Moravek Biochemiquantified by liquid scintillation counting using a I.KB Model 1211
cals, Brea, CA. AFQj, and AFM:. were prepared by reacting AFQi and
beta counter.
AIM, with an equi mo lar amount of trifluoroacetic acid followed by
Instruments. All spectra were recorded on a Perkin-Elmer Model
hydrolysis with water as described previously by Buchi et al. (12). The
650/40 fluorescence spectrophotometer (Norwalk, CT). The synchro
nous scanning technique was performed by interlocking the excitation
Received 7/19/85; revised 3/12/86; accepted 4/4/86.
and emission monochrometers at a wavelength difference (X)and
The costs of publication of this article were defrayed in part by the payment
scanning the sample over a range of wavelengths. The methodology of
of page charges. This article must therefore be hereby marked advertisement in
SFS has been described previously (4-7). Spectral data were collected
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
'To whom requests for reprints should be addressed, at National Cancer
and stored in a Perkin-Elmer Model 3600 data station which also
Institute, Building 37, Room 2C07, Bethesda, MD 20892.
corrected the spectra for variations in xenon lamp output and linearity
2The abbreviations used are: SFS, synchronous fluorescence Spectrophotom
etry; SSC, 0.15 M sodium chloride 0.0015 M sodium citrate, pH 7.0; AFB,-N7during the scan. Limits of detection were defined as the minimum
Gua, 2,3-dihydro-2-(A'7-guanyl)-3-hydroxyafiatoxin
B,; AFBrK'-mGua, 2,3-disignal that was 3-fold above background noise.
hydro-2-(/V7-9-niethylguanyl)-3-hydroxyaflatoxin
BI; AFB,-FAPyr, 2,3-dihydroContour Maps. The data for contour maps were obtained by repeat
2-(7V5-formyl-2',5',6'-triamino-4'-oxo-Ar*-pynmidyl)-3-hydroxyaflatoxin
B(;
edly scanning a sample from an excitation wavelength of 300 to 500
AFBi, aflatoxin B, (other aflatoxins are similarly designated); HPLC, high
nm at different As,typically starting with Xof 10 nm increasing in
pressure liquid chromatography.
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AFLATOXIN

AND SYNCHRONOUS

increments of 4 to 118 nm. Collection of spectral data by multiple

scanning can require up to 3 h/sample. Spectral data were then trans
ferred to the NIH DEC-System-10 computer and analyzed using a
program called MLAB (Modeling Laboratory) which provided the
facilities to view the fluorescence spectra as contours. By projecting the
data onto a 2-dimensional plane as a series of cross-sections of intensity
levels, a map of excitation-emission spectra at multiple AXscan be
viewed in a manner similar to that for topographic maps.

RESULTS
The fluorescence intensity was measured for each of the
aflatoxins over several dilutions to determine the linearity of
fluorescence and the spectral characteristics of the major DNA
adducts and metabolites (Fig. 1). All synchronous scans were
performed at a 5X of 34 nm which is optimal for AFB,-DNA.
Each data point is the average of at least two scans in which
the background fluorescence (nonadducted DNA) was removed
by computer manipulation. A linear relationship exists between
concentration and fluorescence intensity at and above concen
trations of 100 fmol. Below this level linearity diminishes
because of difficult ics in accurately removing the background
fluorescence. A IB, shows a moderate amount of fluorescence
at a Xof 34 nm while AFB2;1,which has a water molecule
substituted across the 2,3 bond in the terminal turan ring, shows
considerably more fluorescence at equal concentrations. AFB,
has been reported to bind to DNA through the oxidative me
tabolism of the 2,3 double bond (14, 16). The increased fluo
rescence from the drivt
i/at onof this bond can be seen in the
standard curve of AFBi-DNA, although its activity is less than
AFBii (Fig. 1). AFBi-N7-Gua has a fluorescence level similar
to that of AFB;
however, AFBrFAPyr, in which the guanine
ring has opened, showed considerably less than the other DNA
adducts and aflatoxin metabolites. All of these metabolites and
adducts showed a fluorescence peak at an excitation wavelength
of 402 nm and could not be distinguished from each other
except by their relative intensity.
AFB,-DNA

10,000

1,000
AFB.-FAPY

100

AFB, N7 GUA

10

100

1,000

10,000

100,000

1,000,000

LOG CONCENTRATION FEMTOMOLES

Fig. 1. Log-log plot of aflatoxin metabolites AFB!, AFH,., and aflatoxin-DNA

adducts AFB,-DNA, AFB,-N7-Gua, and AFB,-FAPyr. Background fluorescence
was removed by computer before plotting.

FLUORIMETRY

Contour Maps of Aflatoxins and Their Metabolites and Ad

ducts. The contour maps shown in Fig. 2 were made by dividing
the spectral data into 20 groups based on the fluorescence
intensity and plotting the groups as a series of dashed lines.
The shortest dashes represent the lowest intensity group and
the longest dashes represent the highest. AFBi and AFBz, (Fig.
2) can be easily distinguished as AFB, shows its peak fluores
cence at 6\ 54 nm and AFBaat SX 38 nm. The differences
between AFB:, and AFBi-DNA are not as obvious because their
peak fluorescence is at similar positions, although the spectral
pattern at lower intensities is noticeably different. Contour
maps of AFMi-DNA are very similar to those for AFBi-DNA.
However, these maps show little resemblance to either AFMi
or AFB], while AFMi is considerably different from AFBi in
2 dimensional scans or contour maps.
Limits of Detection and Optimal Xsfor Aflatoxin Adducts
and Metabolites. Table 1 shows the results of measuring the
various aflatoxin adducts and metabolites at the optimal X
as
determined by contour maps. By scanning the fluorescence of
some aflatoxins at the optimal X,an increase in sensitivity of
detection of 1.5- to 10-fold can be seen over scanning at OX34
nm. The largest increases in detect ahil ity were seen with metab
olites such as aflatoxicol in which the optimal Xof 98 nm
differed significantly from the 6X of 34 nm. The saturation of
the double bond at the 2,3 position of the terminal turan ring
caused an increase in fluorescence of the metabolites. This can
be seen by comparisons of the limits of detection of AFB],
AFMi, AFGi, and AFQ,, with their 2 and 2a metabolites.
In the interests of increasing the fluorescence detectability of
the major product of AFBi with DNA, AFBi-N7-Gua was
further derivati/ed by methylation of position 9 of the guanine
residue. This modification of AFB,-N7-Gua has been suggested
previously to enhance fluorescence (16). As can be seen in Fig.
3, however, methylation of this adduct not only shifted the
optimal Xfrom 34 nm to 86 nm but also decreased the
fluorescence of the chromophore as well as the excitation
wavelength maximum. Thus, the formation AFBi-N7-mGua
was not a viable means to enhance fluorescence.
Resolution of Mixtures Using Three Dimensional Scans.
While contour maps allow discrimination of aflatoxin metabo
lites and DNA adducts in ways not possible with normal syn
chronous fluorescence, their ability to unravel mixtures is lim
ited. An equimolar mixture of AFBi and AFMi have overlap
ping peaks which form a unique pattern. The resolution of the
individual components can be determined by subtracting the
fluorescence of one of the parts, but this depends on knowing
at least one of the metabolites. To solve this problem, we made
use of fourth derivative spectral analysis as described previously
(17). Derivatives of the original spectra are calculated by shift
ing the original spectra by a set wavelength and subtracting this
shifted spectra from the original curve. Repeating this operation
three times results in a fourth derivative of the original spectra.
This procedure emphasizes the individual components of the
peaks and therefore allows overlapping spectra to be differen
tiated. As can be seen in Fig. 4, the components of the mixture
can easily be distinguished following the fourth derivative anal
ysis of the spectral data obtained from the mixture of AFB] and
AFM,.
In Vivo Modification of Rat Liver DNA AFBi. In vi:o AFBimodified rat liver DNA showed a fluorescence spectrum indis
tinguishable from that of in r/m-modified DNA, despite the
differences in AFBrDNA adduct formation. The in vivo DNA
samples had a range of modification of one AFBi residue per
67,000 to one AFB, residue per 15,200,000 nucleotides com-

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AFLATOXIN AND SYNCHRONOUS

118

FLUORIMETRY

\\i
u!/

\ \ '

/ /

V^

\\\\
\ \ \ ! '/ s~
AFBrN7-GUA
82

:-AM
'
t--^1

10
118

<

Aflatoxin M1
82

3
LU

'
10

500 300

EXCITATION WAVELENGTH
Fig. 2. Contour maps and chemical structures of AFB,, AFB&, AFB.-DNA, AFB,-N7-Cua (Adduci I), AFM,, and AFM.-DNA.

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AFLATOXIN

AND SYNCHRONOUS

FLUORIMETRY

Table 1 Optimal 6\for measurement ofaflatoxins and their DNA adducts

of
detection
wavelength
(nm)367402402413340402417389362371398366400415387393395Limit
(fmol)1001001001001001001002502502505005005001,00010,00010,000100,000
SampleAFB2AFBaAFB,-DNAAFGuAflatoxicolAFMj.AFQ*AFB,AFM,AFG2AFB.-N'-GuaAFM,AFG,AFQ,AFB,-diolAFMrDNAAFB,-FAPyrOptimum\7038345498465058669042467062

EXCITATION

b 118

WAVELENGTH

-\\l
//~A\^\\ \
\ vU11//S^\\\\ \ '.

0.7500

AFB1-N7-GUA
DELTA LAMBDA 34NM
AFB1-N7-MeGua
DELTA LAMBDA-86

0.0000
SCO

400

360

CD

NM

300

260

200

EXCITATION WAVELENGTH
400
EXCITATION WAVELENGTH

Fig. 3. Synchronous scans of \l H, V <.iu (0.70 nmol/ml) and Al It, V

mGua (1.04 nmol/ml) at their respective optimal Xs.

pared to the in vifro-modified AFBi-DNA which had a level of

modification of one AI-'H, residue per 20 nucleotides. Table 2
shows a comparison of adduction levels in rat liver calculated
by both fluorescence and radioactivity levels. Aflatoxin-DNA
adducts could easily be detected by fluorescence at doses of 138
and 900 ng/kg. A HPLC profile of the AFB,-DNA adducts
obtained from the 138-Mg/kg dosages is shown in Fig. 5. This
profile reflects the high proportion of AFBi and AFMt imid
azole ring-opened (AFBi-FAPyr and AFMrFAPyr) adducts in
the in vivo DNA samples. This is due to the treatment and
storage of the modified DNA with 10 m M Tris (pH 8.0) to
stabilize the aflatoxin-modified DNA. The in vow-modified
DNA from the 62-/ig/kg-dose animal was not chromatographed
because the specific activity of this dose (58.3 iiCi/nmol) results
in less than 10,000 cpm in the total liver DNA. Comparisons
of the calculated adduct levels in tritium-labeled and non labeled
samples show good reproducibility, but comparisons of levels
by radioactivity and fluorescence show some variance.

EXCITATION WAVELENGTH
Fig. 4. Synchronous scan of an equimolar mixture of 100 pmol of MB, and
! viewed as a 3-dimensional graph (a), a contour map (b), and a 3-dimensional graph of the fourth derivative of the original data (<).
Excitation values are
from 320 to 500 because the first 20 points are lost from each scan during the
computation of the fourth derivative.

Table 2 Measurement ofAFBt-DNA adducts in rat liver

DISCUSSION
AFBj is a mycotoxin produced by Aspergillus flavus and a
potent carcinogen in experimental animals. Al B, is also impli
cated as a risk factor in the etiology of human hepatocellular
carcinoma (17-19). Al H, and its metabolites have been mea
sured by HPLC analysis and immunoassays in body fluids and
tissues from people exposed to AFB in contaminated foods and
beverages (for review, see Ref. 20). AFBi-N7-Gua adducts have

Dose
kg/kg)0

DNAFluorescenceND"0.2

62
138900pmol/mg

0.114.0
10.0
45.0RadioactivityND 78.0HPLCND

ND7.0

58.6
" ND, not detected.
* Aflatoxin-nucleotide adducts measured by high pressure liquid chromatography (see "Materials and Methods").

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AFLATOXIN

AND SYNCHRONOUS

FLUORIMETRY

fluorescent molecules have been used successfully in their quan

titation (25) and thus are presently under study in our labora
tory.

10r

REFERENCES

0.01

Fig. 5. HPLC profile of AFB, metabolite-DNA adducts from liver of rat given
an injection of 138 UKof AFB, per kg body weight.

also been measured in the urine from rats (21) and people (22,
23) exposed to AFB,.
Conventional excitation and emission spectra of aflatoxins
contain many peaks. By scanning excitation and emission syn
chronously, i.e., SFS, with the proper fixed wavelength differ
ence, only one peak emerges. The proper wavelength difference
can be calculated from the 0-0 bands by determining the differ
ence between the longest wavelength band of excitation and the
shortest wavelength band of emission spectra (24). The sensi
tivities of detection of AFB, by HPLC coupled with a fluores
cence detector and by immunoassay are similar.
While SFS is a sensitive method for the detection of aflatox
ins and aflatoxin-DNA adducts (Table 1), it ability to distin
guish between closely related compounds is limited when mea
sured at a single SX.For example, AFB2a differs from AFBi in
the addition of a water molecule across the double bond of the
terminal furan ring, yet synchronous scanning of AFB, and
Al-'Bv, at a Xof 34 nm produces almost identical spectra.
Selecting a o\ to maximize the difference between these metab
olites would result in a loss of sensitivity. By scanning these
compounds at multiple 5Xs, viewing the spectra as contour
maps, and using the fourth derivative spectral analysis, these
metabolites can be differentiated (Figs. 2 and 3). In addition,
contour maps also show the optimum 5X to use for a specific
metabolite or adduct.
The spectral properties of many of the aflatoxin metabolites
and DNA adducts that are known to occur in experimental
animals and humans have been displayed as contour maps
generated by SFS. We have demonstrated that the detection
and quantitation of AFBi-adducted DNA by SFS was compa
rable in sensitivity to that observed using radiolabeled Al B,
and was more sensitive than HPLC coupled with a fluorescence
detector. The limit of SFS detection of AFBi adducts was found
to be one AFBi residue per 1.5 x IO7 nucleotides, which is
similar to that of the ultrasensitive enzyme radioimmunoassay
described previously (11). Preliminary experiments have sug
gested that the level of persistent AFB, adducts in human liver
DNA may be at or below 1 AFBi residue per 1.5 x IO7
nucleotides.3 Thus, the SFS limit of detection of AFBi adducts
must be extended for human studies. Methylation of AFBi-N7Gua did not, however, improve the detectability of this adduct
because it did not increase the fluoresence of this compound as
reported previously (16). Modifications of compounds by highly
3C. C. Harris el ai., unpublished results.

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Measurement of Aflatoxin B1, Its Metabolites, and DNA Adducts

by Synchronous Fluorescence Spectrophotometry
Curtis C. Harris, Gerald LaVeck, John Groopman, et al.
Cancer Res 1986;46:3249-3253.

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