Molecular Dosimetry Cancer researchers are using molecular dosimetry to measure the reactivity of carcinogens with target DNA molecules and to determine whether there are threshold levels of exposure. • • • I l | a r n i n e : The Surgeon • I I I General Has Deter• I m i n e d That Smoking May Be Dangerous To Your Health." But how dangerous? And how much tobacco smoke is enough to cause the variety of cancers, h e a r t disease, and b i r t h defects t h a t well-established epidemiological and animal exposure studies have shown it causes? For the more t h a n 3000 identified compounds in tobacco smoke, as well as for a myriad of environmental and food-related contaminants and toxins, policy makers and public health officials have the unenviable job of trying to figure out, on the basis of these types of studies, the biological r i s k to h u m a n s e x p o s e d to t h e s e compounds. Many compounds, including known toxins a n d carcinogens, produce a wide range of reactions in individuals, and lesions or t u m o r s may not appear until years after an exposure.
FOCUS Some carcinogens interact with each other, some i n i t i a t e t u m o r s , some promote tumors, and some must be metabolized to produce an actively genotoxic form. These complications m a k e it difficult to q u a n t i t a t e the carcinogenicity of a compound by using gross measures of external or int e r n a l e x p o s u r e . " T o t a l body b u r den," or the concentration of toxin t a k e n up by the body, is used as a m e a s u r e of i n t e r n a l exposure. But because of physiological and m e t a bolic differences among individuals and among species, t h e a m o u n t of toxin present does not necessarily reflect how much damage it will cause. Molecular d o s i m e t r y is one way cancer researchers are beginning to quantify the potency of carcinogens and to determine whether there are threshold levels of exposure to them. This technique measures the chemical reactivity of a potential or known carcinogen, mutagen, or other genotoxin with a host "target molecule"—
generally DNA. Most models of carcinogenicity assume that the genotoxin acts by m u t a t i n g or damaging DNA, but in molecular dosimetry some proteins are also examined as easily obtainable indicators with similar reactivity. Many genotoxic agents are electrophilic species that form covalent a d d u c t s w i t h b a s e s along the DNA strand and with certain amino acid residues in proteins. Steven T a n n e n b a u m , of t h e Department of Chemistry and the Division of Toxicology at the Massachusetts I n s t i t u t e of Technology, says, "Molecular dosimetry at MIT really started about 30 years ago with Gerald Wogan's first studies of aflatoxin adduction to DNA." Researchers took what had been discovered in animal e x p e r i m e n t s — t h e observation t h a t the carcinogen formed adducts with DNA and proteins—and tried to use quantification of t h e a d d u c t s as a measure of the effective dose, or the a m o u n t of genotoxin t h a t actually reaches and affects the DNA. "The key to doing molecular dosimetry [effectively] is the use of new analytical techniques t h a t can measure adduct concentrations at the femtomole levels t h a t might be found in people," says Tannenbaum, One advantage of m e a s u r i n g the reactivity of genotoxic agents with DNA is that although physiology and metabolism vary widely among species a n d a m o n g i n d i v i d u a l s , t h e m e c h a n i s m s of DNA r e g u l a t i o n , t r a n s c r i p t i o n , and t r a n s l a t i o n are fairly consistent among vertebrates. Because t h e c h e m i s t r y of purified DNA is even more uniform, it may be more accurate to extrapolate rates of D N A - a d d u c t formation among species than it is to extrapolate rates of tumor formation. Another advantage of molecular dosimetry is that it can be p e r f o r m e d on h u m a n u r i n e or blood samples in vitro r a t h e r t h a n requiring harmful in vivo exposure to potential carcinogens. Tracking down the adducts T a n n e n b a u m ' s group uses GC/MS with negative ion chemical ioniza-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 7, APRIL 1, 1993 · 353 A
FOCUS tion (NICI) or electron i o n i z a t i o n (EI) detection to m e a s u r e t h e a d duct-forming capabilities of aromatic amines and PAHs—particularly re active metabolites of benzo[a]pyrene ( B a P ) — w i t h h e m o g l o b i n (Hb) or DNA. In m a n y studies of h u m a n s , Hb is obtainable in greater quantity than DNA and its adducts have been used as surrogates or biomarkers for DNA a d d u c t f o r m a t i o n a t c r i t i c a l sites, although actual rates of adduct formation for Hb and DNA may dif fer. Hb is found enclosed in red blood cells (RBCs), and its accessibility as a t a r g e t is similar to t h a t of DNA, which is enclosed within the nucleus. To measure the rate of adduct for mation in Hb for known PAHs, Tannenbaum isolates RBCs from h u m a n blood, incubates t h e m w i t h known doses of purified active forms of the carcinogens, washes and lyses them, removes the membranes by centrifugation, and precipitates the Hb (which now presumably contains ad ducts with PAH residues) from the cytosol by adding HC1 and acetone. T h e i s o l a t e d Hb is w a s h e d w i t h 1-butanol to remove free PAHs and sulfur-bonded adducts to glutathione (GSH), a coprecipitating peptide that plays a n important role in scaveng ing toxins from the body and is also studied in molecular dosimetry. The Hb is digested to completion with a nonselective protease, and in dividual amino acids are extracted with ethyl acetate and separated by e i t h e r C 1 8 H P L C or G C / M S . T h e PAHs used are diastereomers of BaP epoxides (the actively genotoxic forms of BaP). When Hb from 1 mL of whole blood is m i x e d w i t h 350 nmol of epoxide, the resulting amino
a c i d - B a P a d d u c t s , isolated in t h e form of diols and tetrols, can be de tected in the single-femtomole range. P a r t of the attraction of molecular dosimetry is that it permits chemists to predict how active a potential car cinogen might be in vivo, although extrapolation of structure-function characteristics cannot be substituted for testing any particular carcinogen experimentally. According to T a n n e n b a u m , simple chemical proper ties such as electrophilicity are bet t e r for p r e d i c t i n g c a r c i n o g e n i c activity of small alkylating agents t h a n of l a r g e r c o m p o u n d s . W i t h bulky carcinogenic agents, steric fac t o r s a n d c h i r a l i t y come into play, a n d it becomes h a r d e r to p r e d i c t genotoxicity from t h e compounds' chemical structures. In addition, the method of hydrolyzing the adducts— acid- or base-catalyzed, or neutral— can affect w h e t h e r t h e adducts are recovered as syn or anti forms. Optimal GC/MS detection m e t h ods vary by the type of carcinogen being tested and by the target mole cule, says Tannenbaum. Most of his group's MS methods were developed for small derivatized adducts with MW < 1000. Because Hb adduct es t e r s with B a P analogues are some what labile, the adducts are deriva tized before G C / M S is performed. Analysis of the adducts by GC/MS is semi-automated for aromatic amines b u t not, as yet, for PAHs, because t h e d e r i v a t i z a t i o n m e t h o d for NICI-MS detection of PAH adducts is trickier. For most P A H - H b a d ducts, NICI-MS results in much less f r a g m e n t a t i o n t h a n does E I - M S . "With NICI, the advantage over EI is in the signal-to-noise ratio, not in
Table 1. Biomarkers for carcinogenicity assessment by molecular dosimetry Biomarker
Matrix
Half-life
Exposure
GSH adduct metabolites
Urine
Hours
Acute
Serum albumin adducts
Peripheral blood
~ 25 days
Short-term
Hemoglobin adducts
Peripheral blood
~ 120 days
Semichronic
DNA adducts
Target tissues
> 120 days
Chronic
DNA adduct repair products
Urine
Chronic
354 A · ANALYTICAL CHEMISTRY, VOL. 65, NO. 7, APRIL 1, 1993
Significance Detoxification or metabolic activation General internal exposure, likely genotoxic Effective dose, likely genotoxic Effective dose, genotoxic Effective dose, possible detoxification with repair
the sensitivity," says Tannenbaum. HPLC with fluorescence linen a r r o w i n g spectroscopy (FLNS) is another adduct detection method be ing used by T a n n e n b a u m ' s group. T a n n e n b a u m says, "We picked u p t h e technique from Gerald Small's group [at Iowa S t a t e U n i v e r s i t y ] . FLNS is comparable in sensitivity to GC/MS, but no one has done a sideb y - s i d e comparison yet." (See also J a n k o w i a k , R.; Small, G. J. Anal. Chem. 1989, 61, 1023 A - 1 0 3 1 A.) T a n n e n b a u m says he is planning a comparative study of the two meth ods in t h e next year and adds t h a t a l t h o u g h F L N S is still p r i m a r i l y qualitative, his group has figured out a way to make it quantitative for Hb adducts. "Real-life" molecular dosimetry M o l e c u l a r d o s i m e t r y c a n also be used to monitor real-life adduct for mation in people who have been ex posed to environmental carcinogens. T a n n e n b a u m ' s group h a s assayed blood samples from smokers for BaPlike a d d u c t s by i s o l a t i n g Hb from RBCs, hydrolyzing it with protease, p a s s i n g t h e d i g e s t s o l u t i o n twice over an immunoaffinity column with a monoclonal antibody ligand raised a g a i n s t PAH adducts, a n d concen t r a t i n g t h e eluted a d d u c t s . O r i g i nally the adducts were analyzed by synchronous fluorescence spectros copy at Δλ = 34 nm, and the amount of p y r e n e - l i k e fluorescent species was calculated by using a trans-anti[14C]BaP-tetrol standard curve. T a n n e n b a u m says his group h a s since abandoned that method for the more accurate GC/MS and FLNS techniques. Typical in vivo environ mental concentrations are 0.2-10 pmol/g of Hb. A l t h o u g h Hb a d d u c t s provide a convenient and a b u n d a n t source of information on genotoxicity, DNA adducts are most closely tied to the initiation of carcinogenesis and mu tagenesis, and they indicate longer exposure to genotoxins. They can be determined in target tissues such as liver, kidney, bone m a r r o w , and g e r m - l i n e cells, b u t fairly invasive procedures m u s t be used to collect the samples. However, products of DNA r e p a i r and excision are often excreted intact in urine and can be determined by several methods. Gerald Wogan, director of the Di vision of Toxicology at MIT, has car ried out small-scale population stud ies in C h i n a and in Gambia, West Africa, to determine the relationship between dietary intake of aflatoxin and formation of adducts to DNA or
to serum albumin, a biomarker for short-term exposure. Aflatoxin, a mycotoxin produced by Aspergillus flavus, can contaminate cereals, peanuts, and other foods. It has been linked to in creased incidences of liver cancer in China and Africa. Wogan's group used monoclonal antibody immunoaffinity chromatography, followed by HPLC with UV or fluorescence detection, to detect D N A - a f l a t o x i n adducts and aflatoxin breakdown products in urine samples. Other DNA adduct detection meth ods include 3 2 P postlabeling, a less instrumentally complex method t h a t is nonselective and has a detection limit of less than one adduct per 10 9 or 10 1 0 nucleotide bases in samples of 1-10 g DNA. The DNA is digested with endonucleases to nucleotide-3'monophosphates and enzymatically radiolabeled with [γ- 32 Ρ]-ΑΤΡ at the 5'-position. The labeled adducts are resolved by four-directional TLC for a fingerprint of adduct nucleotides and quantified by autoradiography. Molecular dosimetry as an epidemiological tool Molecular dosimetry is not the ulti mate measuring tool for carcinogenic risk, says T a n n e n b a u m , but r a t h e r part of an overall scheme of toxic ex posure assessment performed at sev eral levels, each providing a different type of information. The chemical in teraction of a purified known agent with a purified target molecule in an isolated system determines the effec tive dose once the agent has access to the target molecule. U p t a k e and a d d u c t i o n in i n t a c t cells i n d i c a t e some of the localized physiological and metabolic factors t h a t influence the target molecule's exposure to an agent. Whole-body controllede x p o s u r e s t u d i e s in a n i m a l s a r e needed to determine the toxicokinet ics of exposure to an agent, and large population s t u d i e s — m a n y of t h e m morbidity and mortality evalua tions—indicate the outcomes of ex posure. B e c a u s e e a c h level of e x p o s u r e evaluation gives a different piece of information, the in vitro methods of molecular dosimetry are not likely to replace animal exposure studies al together, says Tannenbaum. "Molec ular dosimetry may simplify animal studies—it may even shorten them by helping direct their focus on the most likely a g e n t s and mecha nisms—but ultimately, it's not likely to replace them," he remarks. Still, prospects are good t h a t mo lecular dosimetry will become part of standard regulatory genotoxicity risk
assessment schemes to provide more precise information on the action of agents on h u m a n target molecules. In 1991 the International Agency for Research on Cancer assigned a group to meet in Lyon, France, to evaluate carcinogenic risks. The group con cluded, "As t h e r a n g e of d a t a on mechanisms of action of carcinogens increases, so the set of possible . . . substantive types of evidence avail able . . . will increase. At the same time, inclusion in h u m a n studies of m e a s u r e s r e l a t e d to m e c h a n i s m s
(e.g., molecular dosimetry. . .) will increase the scope and sensitivity of epidemiological r e s e a r c h " (Cancer Res. 1992, 52, 2357-61). Although molecular dosimetry will not replace animal testing or epide miological outcome studies outright, it may change some of the c u r r e n t risk classifications by providing a re fined connection between exposure and genotoxicity and by giving epi demiologists a new molecular tool for assessing carcinogenic risk. Deborah Noble
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