ENVIRONMENTAL C John S. Wishnok Division of Toxicology Massachusetts Institute of Technology Cambridge, MA 02139
The vast majority of substances in the environment—for example, air, water, nutrients, vitamins, naturalproduct anticancer drugs, and insecticides—are benign or actively beneficial. Increasingly, however, there has been a legitimate concern about environmental mutagens and carcinogens. Although useful as a first approximation of potential exposure, the detection or even the quantitation of toxic substances in the environment often is not sufficient for determining individual risk related to the exposure. This is especially true for mutagens or carcinogens. These compounds typically require metabolism, and the resulting active metabolites may act at sites far removed from the site of exposure (e.g., a liver carcinogen may have been absorbed through the skin or inhaled). Rigorous risk assessment requires measurement of the chemical effects resulting from exposure, knowledge of the dose-response relationship, and an understanding of the mechanisms of mutagenesis for each substance being considered. These measurements ultimately depend on the availability of suitable analytical methods, especially for quantitation of the actual active metabolites arising from the e n v i r o n m e n t a l substances. It is particularly challenging to make such measurements in human experiments, because the analyte levels are determined by fortuitous e x p o s u r e a n d t h e s a m p l e s typically are limited to urine or small amounts of blood. Although different classes of mutagens react by specific mechanisms, the majority—after metabolic activation—appear to initiate their effects by reacting at electron-rich sites on DNA. When DNA alkylation and subsequent m u t a t i o n occur, along with a series of subsequent conditions that repair the mutation, car-
cinogenesis may result. The development of sensitive analytical methods and instrumentation over the past two decades, coupled with growing insight into the mechanisms of mutagenesis, has spurred analysts to attempt direct measurements of mutagen adduct formation with DNA or DNA surrogates. Detection and quantitation of these adducts appear to provide the most reliable indication of exposure, because their existence is direct evidence of exposure to active forms of the mutagens; in principal, relative adduct levels may reflect relative risk (2). In this discussion we will be concerned primarily with research carried out at MIT's Division of Toxicology and its forerunners, the De-
1960s that some components of the diet, such as aflatoxins in poorly stored grains (2) and N-nitroso compounds from nitrosation of amines in nitrite-preserved foods (3), might elevate the risk of cancer in humans. Aflatoxins are potent liver carcinogens (2), and N-nitroso compounds constitute a large class of versatile carcinogens for which the potencies and target organs are strongly dependent on the compound's structure (3). These observations led many researchers to assess a variety of nitrite-preserved foods for evidence of n i t r o s a m i n e formation. A l t h o u g h secondary amines are common in food, natural dietary nitrite is rare. Nitrate, however, is plentiful in the
ANALYTICAL APPROACH partment of Applied Biological Scie n c e s a n d t h e D e p a r t m e n t of Nutrition and Pood Science. The focus of much of this research increasingly has been on the direct assessment of individual human exposure to environmental or endogenously formed carcinogens. Two general areas will be emphasized: nitrosamine exposure, including factors involved in endogenous nitrosamine formation, and h u m a n epidemiology via covalent carcinogen-protein adducts in blood. The analytical goal has been to develop methods t h a t can be used routinely to quantitate carcinogenic components of human blood or urine.
N-nitrosamines and nitric oxide In MIT's Department of Nutrition and Food Science and later in the D e p a r t m e n t of Applied Biological Sciences and the Division of Toxicology, the use of MS to detect and monitor m u t a g e n s grew out of earlier work in which MS was used primarily to identify flavoring agents. The shift in emphasis from food science to toxicology was partly the result of discoveries in the 1950s and early
1126 A · ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
diet and can be reduced to nitrite by bacteria, potentially leading to nitrosamine formation (4). Some experiments were carried out to determine if nitrite could react in the mouth or the stomach with co-ingested amines or with endogenously formed amines (5). The realization that low levels of nitrosamines could form in vivo from ingested nitrate and from ingested nitrite increased interest in studying both human and animal nitrate metabolism. A major observation from this research was that nitrate excretion often exceeded nitrate intake, indicating t h a t nitrate was synthesized endogenously (6). The next objectives were to elucidate the biochemical mechanisms of nitrate production and to assess the role of this process in human health risks. Such studies required an analytical system to quantitate nitrate and nitrite in biological media and to study the biochemistry of their formation. Urinary nitrate levels are sufficiently high to allow routine automated quantitation by colorimetry (7). Precursor studies, however, require more subtlety and sensitivity 0003-2700/92/0364-1126A/$03.00/0 © 1992 American Chemical Society
VIVO MONITORING USING G and often have been carried out with GC/MS. A major advantage of MS is t h a t nonradioactive stable isotopomers can be used safely and eth ically in humans. Such experiments typically involve m e a s u r e m e n t of [ 15 N/ 14 N]nitrate ratios in urinary ni t r a t e following a d m i n i s t r a t i o n of [ 15 N]nitrate (8) or labeled precursors such as 1 5 NH 3 (9). The nitrate is derivatized by conversion to nitroben zene, which separates well on polar GC columns (Scheme 1) (7).
compound nitric oxide. When morpholine was added to the culture medium, iV-nitrosomorpholine was p r o d u c e d ; i n t h e p r e s e n c e of [ 15 N] arginine, the nitroso nitrogen in t h e TV-nitrosomorpholine con tained the label. In other words, the nitrosation reaction proceeded t h r o u g h the same i n t e r m e d i a t e — nitric oxide—as the nitrate-produc ing reaction (11). Similar r e s u l t s were obtained in vivo with rats (12). Nitrosamine formation is believed NO,
N0 NO33
HgS HgS 4 4
/T\ + f~\
°
Nitrobe Nitrobenzene: MW=123
5 15, N0
3
+
= \
H 2 S0 4
Following the discovery t h a t ni t r a t e was produced endogenously, the next objective was to define the biochemical details of this process. An a c c i d e n t a l o b s e r v a t i o n , t h a t strikingly elevated nitrate excretion in one volunteer coincided with an intestinal infection, directed atten tion toward t h e i m m u n e system. Subsequently it was discovered that a major cell type responsible for ni trate production was the macrophage (10). 15 N labeling e x p e r i m e n t s w i t h macrophage-like cell lines in culture demonstrated that the nitrogen source was one of the two equivalent guanido nitrogens of L-arginine and that the major intermediate in the synthesis was the simple inorganic
Scheme 2
N0 2
0
[15N] nitrobenzene: MW = 124
Scheme 1
[15N] arginine - ^ -
15,
15
N0 + 0 2
to proceed via the oxidation of NO to N 0 2 and then N 2 0 3 as well as (to some extent) N 2 0 4 , which can react with secondary amines to yield Nnitrosamines at a physiological pH (13). These transformations are sum marized in Scheme 2. In addition to the formation of ./V-nitroso compounds, NO may dam age DNA via nitrosative deamination of nucleic acids in the presence of 0 2 . These alterations in DNA could lead to mutations t h a t are important in activating human oncogenes (14, 15). The relevance of these pathways to humans was demonstrated by an ex periment in which two volunteers in gested [ 15 N]arginine. Two individu als consumed low-nitrate diets for two days. Urine was collected for two
15
Table I. Nitrate and [15N]nitrate excretion in humans 15 Subject Urinary N0 3 " Urinary NOj
Ν
^ΦN203
24-h periods. At the beginning of the second 24-h period, each drank a so lution containing labeled arginine. Increased levels of [ 1 5 N]nitrate were detected in urine samples collected the next day (Table I [16]). The top numbers were obtained from urine analyses from the first 24-h period (baseline levels). The bottom num bers were obtained from the second 24-h period. During and after these studies, a s t a r t l i n g n u m b e r of e n d o g e n o u s sources and functions of nitric oxide were discovered. In addition to NO synthesis by stimulated macrophages, NO production by endothelial cells (as endothelial-derived relaxing fac tor, EDRF) activates a heme-depen dent guanylate cyclase, which leads to vascular smooth muscle relax ation. The role of NO in intracellular signaling is known to occur across species lines in cells from m a n y sources, including placental and pul monary vessels, and in the perfused heart. NO is also produced by hepatocytes, neutrophils, and cells in the brain, where it functions as a neuro transmitter. These topics are sum marized and discussed in several ex tensive and complementary reviews (17-19). Most analyses of NO production have been indirect; that is, they have involved quantitation of ultimate nitrite/nitrate concentrations (20) or measurement of related physiological effects such as muscle relaxation (21). Direct m e a s u r e m e n t s of NO have been carried out with thermal
°3
A
650830
Β
1770 828
^
0 24 0 17
Source: Reference 16
^ ^ *
15
[ N] nitroso compounds
a
μπιοΐββ/όβν
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992 · 1127 A
ANALYTICAL APPROACH energy analysis (TEA) (21) or with GC/MS (22). In a recent paper we de scribe a membrane mass spectrome ter inlet for direct NO quantitation in either gases or liquids that can monitor NO production at concentra tions as low as 1.4 μΜ in real time (23). F i g u r e 1 s h o w s t h e d o s e response characteristics of the inlet in both phases. A recent electrochem ical microprobe method appears to have greater sensitivity (24).
Quantitation of chemically altered DNA in individuals is a challenge be cause of problems such as low adduct levels; efficient repair of the damage; dilution of adduct levels by cell divi sion; and the difficulty of sampling human DNA, especially in the wide variety of target organs. Stimulated largely by suggestions from Lars Ehrenberg and co-workers (25), many cancer epidemiologists have focused attention on protein ad ducts with the assumption that lev els of c a r c i n o g e n - p r o t e i n adducts will reflect levels of carcinogen-DNA adducts. In other words, the electrophilic intermediates that react with nucleophilic sites on DNA will also
Mutagen-protein adducts The formation of altered DNA by an active carcinogen metabolite is a principal event in the complex pro cesses leading to clinical cancer.
5000-
4000
