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331 IV. PHYSIOLOGICAL FLUIDS STUDIEy: MTAGEPdICITY AND PROFILING Pd. P. P•faskarinec, R. W. Harvey, and T. K. Rao* In addition to the development of methodologies for the analysis of nicotine/cotinine in serum and urine, we have been examining methods to determine the efficacy of the Ames bacterial mutagenicity assay and high resolution profiling of physiological fluids in assessment of the biochemical impact of chronic smoke exposure on experimental animals. The ultimate objec- tive of these studies is the identification of biochemical "markers" indicative of early tendencies toward pathology. Such markers could then be used to develop less hazardous cigarettes or to alter smoking patterns on an individual basis. Here we report on progress made in scoping studies in two distinct areas. P4utac.Leniclty. The Ames test for bacterial mutagenicity has come to be considered as a relatively reliable indicator of the presence of mutagenic substances. Compounds found to be mutagenic are often carcinogenic. It has been shown that human smokers may have mutagenic constituents in their urine (1). Because there exist suspected correlations between bladder cancer and cigarette smoking, we have performed pilot mutagenicity screening studies on urine samples from beagle dogs in the chronic exposure at Borriston Laboratories and from baboons in the inhalation studies at SWRI. The urine (100 ml) is passed through an Amberlite XAD-2 column, and the retained organics are eluted with acetone. The acetone is evaporated, the residue taken up in DMSO and plated on the Petri dishes for bioassay (Ames/Salmonella system). In initial work, it was determined that the TA-98 Salmonella strain would be the most suitable for studies of mutagens in urine. This strain is susceptible to frameshift mutations of a non-specific nature. Consequently, Arochlor-activated rat-liver homogenates were used in conjunction with the TA-98 strain. *Biology Division.

332 A series of urine samples from the dogs at BRL were subjected to biological testing with this system. Results of the bioassays on two animals per group are presented on Table III-4.,_, Interestingly, the Code 32 exposure group animals showed no significant urine mutagenicity. This may be due to the fact that these samples were much more dilute than the others, and a subsequent test of Code 32 dog urines has shown activity intermediate to that of the Code 13 and Code 11 animals. A larger group of animals have been sampled and are currently being tested. In addition, this approach has been applied to a series of urines from smoking baboons. Results of the mutagenicity screening are given on Table 111-5. The most important finding was that of a positive control. That a sham smoked animal has highly mutagenic urine may suggest that the animal has much different metabolic pathways, an "individual susceptibility". Identification of the active components in the urine might provide some insight into genetic pre- disposition to cancer. These samples will be characterized during the coming year for the purpose of identifying the mutagenic constituents. i•lulticom~aonent Profiling and Scoping Studies. A second major effort has been the multicomponent analysis of physiological fluids. The aim of these studies has been the detection of early changes in the biochemistry of the smoke insulted organism which would be characteristic of developing pathology. These investigations are still in a preliminary stage, and the major progress to date has been the scoping of methodologies which appear promising from a cost-effective standpoint. Approaches which appear promising include: (1) Urine Volatiles: The technique involves purging a 1 ml urine sample with 1500 cc of helium through a trapping system filled with an adsorptive porous polymer, Tenax-GC. The adsorbed com- ponents are then thermally desorbed onto a glass capillary GC column for analysis. Comparison of representative profiles from

TABLE 111-4 Mutagenicity of Dog* Urine Exposure Group Activity rev m} Description Piate 35 20 u1 50 il1 Sham 46 68 Not Active Code 11 80 145 Highly Active Code 13 70 94 Statistically Code 32 50 61 Significant Activity Not Active Code 67 50 58 Not Active *Dog urine samples courtesy of Dr. Patricia Buhl, Borriston Research Laboratories. L994E468

334 TABLE 111-5 Mutagenicity of Baboon Urine* Animal Number Subjective Assessment of ~ Smoking Mutagenicity X-711 Sham X-712 Sham +++ B-734 "Old Pro" (Good Smoker) + X-773 Bad Smoker (Takes Short Puffs) ++ X-774 Bad Smoker (Avoids Smoke) + *Baboon urine samples courtesy of Dr. Walter Rogers, SWRI.

335 urine of sham and smoke exposed beagle dogs is made on Figure 111-6. There are important qualitative differences between the profiles. Al- though no quantitative conclusions can be made at this time, the results have demonstrated that profiles obtained from the urine of control animals are significantly different from those obtained from the urine of smoke exposed animals. Since individual variations within given exposure groups were not great, significant results should be obtain- able from a relatively well-controlled, small set of samples. Compounds which have been identified in urinary volatiles profiles include (3) ketones, aldehydes, alcohols, furans (carbohydrate metabolites), various pyrazines and pyroles (amino acid metabolites), terpenes, alkyl-sub- stituted benzenes, phenols and cresols, isothiocyanates. Many of these are possible smoke constituents. Profiles of urinary volatiles should provide information of both a metabolic and smoke marker nature, and thus may be useful for dosimetric and impact assessment purposes. For example, with similar profiling methods, 4-heptanone has been detected in the urine of diabetics which may indicate a predisposition toward this disease. It is likely that early indicators of cigarette smoke-induced pathology will be found in the profiles of smoke-exposed animals. (2) Multicomponent Steroid Determination in Physiological Fluids: The steroid hormones are responsible for control of a wide variety of meta- bolic activities. Cortisol controls gluconeogenesis, increases protein catabolism, suppresses lymphatic tissue growth, is antiinflammatory and yet ulcerogenic. Cortisol levels are good indicators of stress. Al- dosterone is responsible for whole body electrolyte balance. The sex hormones, androgens and estrogens, influence vascularization, red blood cell count, blood hemoglobin content, and stimulate sebacious gland se- cretion. Thus any alterations in steroid biosynthesis or metabolism due

FIGURE 111-6 URINARY VOLATILES PROFILES: COMPARISON OF SFNAN AND SMOKE EXPOSED BEAGLE DOGS ORNL-OWO 78-1636 (0) Sham 1 (b) Code 11 Exposure Group 4q LkjPbkJ l I I I I I I I 40 60 80 100 120 140 160 180 TEMPERATURE (°C) € € € € L € € c € € ~ L 0 10 20 30 40 50 60 70 80 90 100 110 TIME (min) 4GB41=B-

337 (3) cigarette smoke exposure would produce great perturbation on the organism. Furthermore, many known promoters and carcinogens have chemical structures similar to the steroids (e.g. TPA, MCA). While the precise effect of the steroid system on the carcinogenic process are not yet understood, much epidemiological data implicates the steroids in the process. Adrenocort- ical activity decreases with increasing age, while likelihood of cancer increases. Marked sexual differences are seen in many epidemiological studies of various cancers. Androgen/estrogen ratios have been implicated in various cancers. The complex nature of the steroids themselves, as well as the lack of understanding of steroid-cell interaction, has impeded efforts to clarify the role of the steroids in the carcinogenic process. However, recent advances in analytical chemistry of the steroids have permitted the multicomponent determination of steroids in body fluids (2). This methodology has been applied to the study of steroids in the urine of smoke exposed dogs. The results have indicated that such profiles can be reproducibly obtained, and that certain endocrine effects of smoke exposure can be visualized. More detailed studies are indicated, in- cluding plasma and urinary steroid structural identification. Plasma Cortisol: Cortisol levels in a few selected animals from the Borriston inhalation study have been measured by radioimmunoassay in order to evaluate the effects of smoke exposure on the levels of this important hormone and to assess the potentially stressful nature of the exposure. The data suggest that only dogs exposed to the Code 67 vari- ant (phenyl-methyl oxadiazole containing) were under significant stress. Stress can have an important effect on metabolic processes. Since the

338 impact of the noxious material in cigarette smoke may be expected to induce a stress-type reaction in naive smoking animals, it would seem of great interest to monitor plasma cortisol levels for all animals in a bioassay over the course of the exposures. (4) Urinary ~-Glucuronidase Levels: One of the mechanisms which has been proposed for bladder cancer involves the affinity of potentially car- cinogenic organic molecules for the bladder cell lipid layers. Normally, organic molecules are easily excreted from the bladder as conjugates with hydrophilic species. f3-glucuronidase is responsible for deconjugating (and thus reactivating) excreted organic molecules in the bladder. The data involving mutagenicity in urine of smoke exposed animals led us to investigate the possibility that levels of this enzyme might be sufficient to partially account for the presence of mutagenic (potentially carcino- genic) substances in the urine. The results of these preliminary studies are given in Table 111-6. The observed levels agree well with the muta- genicity data. Further studies are indicated to determine if ~-giucuroni- dase levels correlate with mutagenicity over a wide range of smoke exposed animals.

TABLE 111-6 S-Glucuronidase Activity in Urine of Dogs Exposed to Cigarette Smoke Dog Number Cigarette Code Units ~-Clucuronidase 5205 Sham 1.5 5217 Sham .2 5289 11 5.6 5313 11 4.2 5096 13 4.6 5075 32 .8 6021 67 2.0 6023 67 5.2 E694C469

340 References a, 1. "Concentration of Mutagens from Urine by Adsorption with the No,n~polar Resin XAD-2: Cigarette Smokers Have Mutagenic Urine," E. Yamasal~l and ' 1. Acad. Sci. USA, 74, 3555 (1977). ~ B. N. Ames, Proc. Nat 2. "Analysis of Steroids by Glass Capillary Gas Chromatography Mas's ~ Spectronietry: Applications to Cannabis Pharmacology," M. P. Mask4 rine Ph.D. Thesis, Indiana University, 1977. 3. "Concentration and Analysis of Volatile Urinary Metabolites," Zlatkis, et al., J. Chrom. Sci., 11, 299 (1973). A. ~