Journal of Nuclear and Radiochemical Sciences, Vol. 2, No. 1-2, pp. R9-R12, 2001

Reviews
Applications of 14C-AMS for Carcinogen, Pharmaceutical and Nutrient Studies
Wang Haifang, Liu Yuanfang*, and Sun Hongfang
Department of Applied Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing
100871, China
Received: November 2, 2001; In Final Form: November 16, 2001
Accelerator mass spectrometry (AMS) is an ultrasensitive measure for tracing 14C labeled molecules in vivo or
detecting the biomarker for assessment of carcinogenesis. Basic principles, wide applications and new progressesof bio-14C- AMS have been presented. It is a state-of-the-art tool for measuring the adduction of biological molecules with xenobiotics, including carcinogens, drugs, agrochemicals, nicotine etc.. We have studied the genotoxicity and proteins adduction of smoking specific nicotine and its nitrosamine derivative NNK since 1994. The
successful applications have proven the effectiveness of AMS in assessment of cancer risk, screening of drug toxicity and studies of nutrient uptake. In particular, AMS is characterized by measuring xenobiotics at very low doselevels relevant to human environmental exposure. It is sensitive and precise to an attomole (10−18 mole) of 14C per
mg carbon. Although it has some shortcomings, undoubtedly, AMS possesses an evident merit of high sensitivity
and will have widespread applications in the biomedical sciences.

1. Introduction
AMS is usually built up based on a tandem Van de Graaff
accelerator or other specific accelerator, which consists of ion
source, injection system, acceleration facility, high-energy
analysis system, detectors and computer controlled system. In
general, AMS quantitates long-lived isotopes by counting
isotopic nuclei directly through a variety of physical instrumentations.1 It provides much greater sensitivity for isotope
detection than other decay counting methods, e.g., liquid scintillation counting (LSC). The sensitivity is at attomol of 14C
per mg carbon. The detection limit of DNA adducts in AMS
measurement reaches 1 adduct/1012 nucleotides.2 Besides thevery high sensitivity, the merits of AMS also include smaller
sample size, lower specific activity and less measuring timeallowed in the measurement.
At present, the major application of AMS is in the fields ofgeoscience, oceanography and archeology based on thechronological evaluation and dating principle. The earlypioneering work of bio-AMS (AMS in biomedical sciences)
was done by LLNL in 1989.3 They measured adduction of
DNA with a food carcinogen MeIQx in mouse liver by AMS.
In 1994 Liu et al.4 published a review paper on the non-dating
applications of AMS, including various biomedical applications. Nowadays, a variety of studies in bio-AMS field havebeen done and this sophisticated technique has drawn much
more attentions in the biomedical community.
The long-lived isotopes can be used in bio-AMS studies are
14
C, 3H, 26Al, 36 Cl, 41Ca, 99Tc, 129I etc.. However, the moststudies have been carried out with isotope 14C, an isotope ofthe most essential element in the living organisms. This
review paper deals with the studies of carcinogens, pharmaceuticals and nutrients using 14C- AMS.
2. Carcinogens
The carcinogenesis of a xenobiotic is a function of the dosedelivered to an organ, as the dose remains in the tissue
inducing the adduction or affinity of the xenobiotic to a key*Corresponding author. E-mail: ylin@pku.edu.cn.

biomolecule. The reaction of chemical xenobiotic with DNA
appears to be one of the most important and easily happened
events in the initiation phase of cancer. The nature of bindingis mainly forming a covalent bond between the small toxicant
molecule and the macromolecule. Also there are weak noncovalent bindings, such as groove interaction and electrostaticinteraction around DNA helix. Thus, a dose and a time
response curve for each organ are required for assessing thecancer risk of organs damaged from a xenobiotic. In the past,
the relationship between adduct formation and exposure wasprimarily established at high dose levels of carcinogen due tothe limitation of sensitivity in assay. Bio-AMS may offer adistinct advantage of detecting extremely small amounts of
14
C-carcinogen bound to DNA.
Table 1 shows 14 C-AMS study of carcinogens either tohuman or animal by researchers of Lawrence Livermore
National Lab (LLNL) and their collaborators in U.S. or U.K..
MeIQx, a hetrocyclic amine carcinogen found at the ppb levelin cooked beef, is the first xenobiotic studied by AMS. 3 A
linear relationship of DNA-MeIQx adducts and doses over arange of 500 ng/kg b.w. (body weight) to 5 mg/kg b.w. inmouse liver was observed. The 500 ng/kg b.w. dose level,
corresponding to 1 adduct/1011 nucleotides, is the lowest dose
the authors can statistically discriminate from the control. Thisis ten to hundred folds improvement over the best sensitivity
offered by the 32P-post-labeling assay, and 3 to 5 orders ofmagnitude better than other techniques used for quantitative
assay of DNA adducts.
TCDD (Dioxin) is an extremely toxic and potent
carcinogen. Particularly, in recent years it was recognized as anotorious carcinogenic toxin. In the past, TCDD-DNA
adducts were not detectable following 6 months of repeatedweekly high dose exposure using the most sensitive assay
available at that time.5 Because of very high-dose taken in theprevious work the extrapolation of the effects to lower relevant
exposures was suspected. Compatibly, the present AMS
measurement has showed that no TCDD related adducts were
found except for very small amounts of adduct observed at thelethal dose level. 6 As a very rare exception, these results
suggest that the potent carcinogenesis of TCDD is not originated from the effects of initial DNA adduction.
We have measured DNA adduction with one major nitrosa-© 2001 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 12/28/2001

Haifang

R10 J. Nucl. Radiochem. Sci., Vol. 2, No. 1-2, 2001
TABLE 1: Carcinogens Studied by 14C-AMS
Carcinogen

Biomolecular research

MeIQx, 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline

DNA adducts in mouse liver3
DNA adducts in rat liver and distribution9
Hemoglobin (Hb), albumin adducts in rat and human10
Metabolism in human urine11
Distribution and DNA adducts in human and rodent colon12
DNA adducts in rodent and human colon13

TCDD, 2,3,7,8-tetraChlorodibenzo-p-dioxin

DNA adducts in mouse liver, not observed6

PhIP, 2-amino-1-methyl6-phenylimidazo[4,5-b]
pyridine

DNA adducts in mouse liver, lung, heart and metabolites; fate and distribution in mouse14
Distribution and metabolism in female rats and their pups15
DNA and protein adducts in human colon and blood16
Colon DNA adducts and metabolites in urine of human and rat17

BenzeneDNA and protein adducts in mouse mallow chromosome; distribution and liver DNA adducts inmouse18
DNA adducts in liver and mallow of rat and mouse19

Trichloroethylene

DNA and protein adducts in mouse liver20
Initial uptake kinetics in human skin21

NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

DNA adduct in mouse liver7
Decay kinetics of adducts8

tion derivative of nicotine, 4-(methylnitrosamino-)-1-(3pyridyl-)-1-butanone (NNK). It is a well-known potent andpredominant carcinogen and widely present in cigarette smoke
as well as unburned tobacco. Its content in cigarettes isroughly 1/10,000 of nicotine content. A good linear dependence of DNA-NNK adduct number on the exposure doselevels is shown.7 The decay kinetics of DNA-NNK adducts
measured shows a slow decay process.8
3. Carcinogenic Suspects
It is very important to identify whether an organic
compound is carcinogenic or not in the cancer toxicologicalstudy. In particular, some organic compounds have proven no
property of carcinogenesis by conventional medical tests, butthey could be genotoxic that indicates they could finallybecome carcinogenic. AMS has shown a greater potential thanthe conventional medical methods to solve the identification
problem.
3.1. Nicotine. Nicotine, 3-(1-methyl-2-pyrrolidinyl)-pyridine is a major alkaloid in tobacco products, typically comprising 1-2 % weight of tobacco. Nicotine related to smoking is adominant factor for tobacco addiction. So far it is unclear
whether it is carcinogenic to humans.
Since 1994, our group has carried out a series of genotoxicity studies of nicotine in mouse by 14 C-AMS.7, 8, 22−24 We
found that mouse liver/lung DNA adduction as well as
histones(H1 & H3)-nicotine adduction increased withincreasing dose level of nicotine, which was equivalent to thelevel of human exposure to cigarette smoking. Very good
linear correlations in the dose response were given. Our findings demonstrate clearly that nicotine damages DNA as well as
its closely related significant nucleoprotein histones throughthe chemical binding in-between. Besides, we have measured
the decay kinetics of the adducts and the effect of NaNO2 onliver DNA adduction. By these 14 C-AMS studies we havedrawn to an important point that nicotine should be no longer
only recognized as an addiction factor in smoking, but also a
potent carcinogen per se.
3.2. Insecticides and Herbicides. We began studying the
genotoxicity of insecticide pirimicarb by AMS early in 1994.25

Pirimicarb is a selective and effective antiaphid insecticide
widely used in China and other tens countries. By using AMS
we found DNA adduction with pirimicarb in mouse liver after
exposure to environmental dose of pirimicarb. DNA adducts
increased linearly with increasing exposure doses in a log/log
plot. However, the number of adducts greatly reduced as thesurvival time elapsed. In another word, pirimicarb is an insecticide of low genotoxicity, probably owing to the potent repair
mechanism occurred in the damaged DNA.
Other groups also studied insecticides exposed in environment, such as atrazine (triazine herbicide), which has widespread use in fields with low toxicity to mammals. The
experiment was designed to examine metabolites of atrazine inhuman urine by AMS.26 A mean excretion half-life of atrazine
in urine of 24 ± 3 h was measured by AMS.
As a special case, the aim of studying the uptake decrease ofpyrethroid insecticide 14C-permethrin, which is taken as ananalogue of a warfare toxin, in rat central nervous system(CNS) using AMS is to evaluate the medication effect of apyridostigmine, a pretreatment nerve agent for “Gulf War
Syndrome”.27
3.3. Therapeutic Drugs. Tamoxifen, Z-2-[4-(1,2-diphenyl1-butenyl)-phenoxy]-N,N-dimethylethanamine, is widely usedin adjuvant therapy of breast cancer as a cancer chemopreventative agent. By AMS study carried out at LLNL, tamoxifen
was shown to bind to liver DNA of female rats in a linear
dose-dependent relationship over a range of 0.1-1 mg/kg b. w.,
compatible with the therapeutic dose used in women (20mg/person per day).28 The genotoxicity of analog of tamoxifen, toremifene (Z-2-[(4-chloro-1,2-diphenyl-butenyl)
phenoxy]-N,N-dimethylethanamine) was also measured by
AMS.28 Toremifene binding to rat liver DNA was one order ofmagnitude lower than that seen with tamoxifen. These results
remarkably show that some effective anticancer drugs might
be genotoxic per se, thus, probably becoming carcinogenic andinjurious towards the cancer patients administered drug.
4. Pharmaceuticals
In the pharmaceutical tracing studies the quantitation of 14Cradioactivity is usually made by decay counting with a liquidApplications of 14C-AMS for Carcinogen

J. Nucl. Radiochem. Sci., Vol. 2, No. 1-2, 2001 R11scintillation counter (LSC). Compared with LSC, AMS has alarge attraction for its much higher sensitivity, in 105−106 foldenhancement.26 Consequently, the radioactivity as well as the
sample size required for AMS is much smaller than that for
LSC. Garner et al.29 of the CBAMS of York, UK have made avalidation study comparing AMS with LSC measurements. In
this experiment they used human plasma in vitro spiked with
14
C-fluconazole, an antifungal drug. An excellent correlation
between the two methods (correlation coefficient 0.999) was
resulted.
14
C-AMS has shown the great potential in studies of absorption, distribution, metabolism and excretion (ADME) of pharmaceuticals in human. It is surely recognized by the medical
scientists that ADME studies in humans are absolutely indispensable for developing a new drug before it enters the phaseof clinical trials in hospital.
Garner et al. 29 also have analyzed the pharmacokinetic
excretion of drug X (anonymous for the drug company’scommercial profit) in human volunteers administered orally
300 nCi of 14C-labelled drug X. Each tablet contained 30 mg
of cold drug. This dose was selected as it equated to 0.9 µSv
i.e., below the British official regulatory standard. Percentage
excreted of drug X in total faeces and urine were measured interms of mass balances. The authors believe that this kind of
early clinical studies by using AMS can shorten largely the
drug development times, e.g. 1 yr, thus ensuring that drugs
come to market earlier.
We are planning to conduct the ADME studies of the bioactive extracts of some Chinese medicinal herbs using AMS
technique. These studies may find access to the discovery of
the receptor targets of the extract molecules in humans.

6.1. AMS Tests in Human Subjects. LLNL began thestudies of uptake and metabolism of anticancer drugs in cancer
patients early in 1990.6 Nevertheless, even though the 14C
radioactive dose administrated to human host was very low, farapart from the radiation limit standard, it is hard to recruit
testing patients or volunteers. In the preceding paragraphs, we
presented the fat metabolism studies in humans by Swedishgroup and the metabolism studies of folic acid and β-carotene
in volunteers by LLNL group. 3 0 , 3 2 In 1999 LLNL alsomeasured DNA-MeIQx adducts and DNA/protein-PhIP
adducts in 5 colon-cancer patients. 16 In all the tests, theradioactivity of 14C was limited to only a max. < 1 µCi administered per person. The poor availability of the testing bodies
is an inevitable bottleneck of this study.
6.2. AMS Coupled with HPLC and Other Techniques.
High performance liquid chromatography (HPLC) is a popular
technique for the chemical separation of complex mixtures,
whose profile can afford information of chemical mechanismand estimation of the biomedical pathways of metabolism.
Compounds elute from the HPLC systems with characteristics
retention times and can be collected in fractions, which can be
subsequently graphitized for AMS measurement.34 A combination of AMS with this sophisticated method has largely
benefited the studies of xenobiotics PhIP, trichloroethylene,
benzene, atrazine, β-carotene, drug X etc..
Besides, gas chromatography (GC), thin layer chromatography (TLC) and radioimmunoassay have also been coupledwith AMS that also makes AMS more efficient and satisfactory in the biomedical applications.

5. Nutrients

7. Prospect of Bio-14C- AMS Studies

Around 1998 Vogel et al.30 created a new application fieldof AMS studying the distribution and metabolism of nutrients,
some vitamins as folic acid and β-carotene. These studies
could be largely extended to a great deal of other common
nutritives, which are very important to ensure the healthy lifeof humans. In the experiments, 100−200 nCi doses of 14C asmolecular labels in nutrients were given to the testing volunteers without any radiation damage. In fact, a 70 kg person
contains ~ 90 nCi of naturally produced 14C in body. A 100
nCi dose yields at least a 20:1 signal to background level for
tracing labelled compounds that may provide satisfactory AMS
measurements.31
Since the 14 C-triolein, glycerol tri[1- 14 C]oleate tests arewidely used in the clinical medicine for the determination of
fat malabsorption, AMS has been applied in the long-term
turnover study of this fatty acid ester in humans at Universityof Lund, Sweden.32 The measurements were carried out up to
4.5 yr. after administration. 14C contents in exhaled air, fat,muscles, bones, faeces and urine were measured. 60% 14C
activity was recovered and the major part of the missing fraction was owing to the uncertainties connected to the large
amount of the endogenous production of CO2.
Very recently, AMS was used in an exotic way to distinguish fraudulent synthetics from the naturally products.33
Nowadays, the public consumer trend towards foods andbeverages labelled “ natural ” has created a growing demand.
However, in the market so many “natural” foods are blended
with inexpensive synthetics. The source of synthetics ismostly petrochemically derived compounds, which contain a
low level 14 C from the “dead carbon”. Through the AMS
measurement it is easy to find the fake nutrient blended in thereal natural products which originally contains a higher levelof 14C equilibrated with the ambient 14C in the natural environment.

Bio- 14 C-AMS offers the quantitative measurement withultra-high sensitivity of low-frequency biomolecular events
following exposure to environmental dose level of xenobiotics.
Samples of 1 mg or less are easily handled and absolute resolution can be 1 attomole per mg of carbon that has priorities over
all the other methods. Of course, it has been a new advanced
tool in the biomedical, toxicological, pharmaceutical and nutritional sciences. Particularly, it will become increasingly
important in cancer risk assessment, pharmaceutical screening
and ADME studies of drugs and nutrients.
However, there are some remarkable disadvantages or limitations in AMS method: the very large size and high investment of accelerator, the special radiolabelled compound andthe troublesome preparation procedure of the graphite sample
all required for AMS measurement. Moreover, the large
community of biomedical researchers is largely unaware ofthis new technology and the term AMS is often a confusing
glossary to them.
But we believe that the merits and advantages of AMS
method far outweigh its defects and disadvantages. The
researchers of AMS are gearing up to meet the foreseeing
needs of the biomedical sciences.

6. Important Progresses of Bio-14C- AMS

Acknowledgement. This work was supported by the
National Natural Science Foundation of China (Grant Nos.
19935020 and 29871004).
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