Vol. 1, /153-1163, October /995 Clinical Cancer Research 1153
Identification of a Class 3 Aldehyde Dehydrogenase in Human Saliva and Increased Levels of This Enzyme, Glutathione S-Transferases, and DT-Diaphorase in the Saliva of Subjects Who Continually Ingest Large Quantities of Coffee or Broccoli’
Lakshmaiah Sreerama, Matthew W. Hedge, and achieve its full therapeutic potential would be selected based Norman E. Sladek2 on whether high or low enzyme activity would be favorable in that regard. Such measurements may also be useful as an Department of Pharmacology, University of Minnesota Medical indicator when exposure to carcinogenic/teratogenic/other- School, Minneapolis, Minnesota 55455 wise toxic environmental/industrial/dietary agents that in- duce these enzymes is suspected. ABSTRACT Human saliva was tested for the presence of cytosobic INTRODUCTION cbass 3 abdehyde dehydrogenase, gbutathione S-transferases Celbuban expression of ALDH-33 and certain other enzymes a, ,i, and IT, and DT-diaphorase, enzymes that are known to (e.g., glutathione S-transfenases and DT-diaphorase) can be catalyze the biotransformation of many xenobiotics, includ- markedly and coordinately increased by both monofunctional ing some that are carcinogens and some that are antineo- (e.g. , phenolic antioxidants such as catechob, hydnoquinone, and pbastic agents. Each of these enzymes was found to be 2,6-di-tert-butyl-4-hydnoxytobuene) and bifunctional (e.g., present in this fluid. Inducers of these enzymes are known to 3-methybcholanthnene and 3,4-benzpymene) inducems4 (1-3). In- be abundantly present in the human diet, especially in cer- duction of glutathione S-tnansfenases and DT-diaphorase is tam vegetables and fruits. Further investigation revealed viewed with particular interest because these enzymes are that the salivary content of these enzymes rapidly, coordi- known to detoxify certain carcinogens (4). Inducers of these nately, and markedly increased upon daily consumption of enzymes are abundantly present in certain components of the relatively large amounts of coffee or broccobi. The enzyme human diet, e.g. , members of the Cruciferae and Liliaceae activities of interest rapidly returned to basal bevels when families of vegetables (5, 6). Certain food additives (e.g., 2,3- these substances were removed from the diet. Given the tert-butyl-4-hydmoxyanisobe) and pharmaceuticals (e.g., olti- important role that cytosolic class 3 abdehyde dehydroge- praz) also act as inducens of these enzymes (reviewed in Refs. nase, the glutathione S-transferases, and DT-diaphorase are 7-9). Vegetables, fruits, and chemicals that induce these en- thought to pbay in determining the carcinogenic potential of zymes prevent experimental cancinogenesis, i.e., they effect a some cancer-producing agents as well as the cytotoxic po- chemopreventive action, and the former is thought to be caus- tentiab of some antineoplastic agents, and assuming that ative, at beast in part, of the batten (reviewed in Refs. 7-9). their salivary levels reflect their tissue bevels, quantification Chemoprevention mediated in this manner is an immenseby of the salivary content of one or more of these enzymes, a attractive idea for obvious reasons, and clinical evaluation of noninvasive and relatively easy undertaking, could be useful oltipnaz in that regard has already been initiated (10-12). De- in: (a) preliminarily assessing the chemopreventive potential sirabbe in such investigations is an estimate of relevant enzyme of various diets and drugs; (b) establishing the optimal dose induction. One way of doing this might be to quantify the and schedule in Phase I clinical trials for any putatively relevant enzyme activities in serum, peripheral blood bympho- chemopreventive diets or drugs of interest; and (c) the ra- cytes, on tissue biopsies (13-15). Limitations of these ap- tional selection and use of chemotherapeutic agents, since several are inactivated, and a few are activated, by these enzymes; alternatively, the antineoplastic agent could be selected first and then a diet that enables the agent to 3 The abbreviations used are: ALDH-3, human cytosolic class 3 alde- hyde dehydrogenase; mIU, milli-International Unit of enzyme activity [nmol NAD(P)H formed/mm in the case of aldehyde dehydrogenase activity, nmol of the conjugate of 1-chloro-2,4-dinitrobenzene and glu- Received 1/20/95; revised 5/15/95; accepted 5/18/95. tathione formed/mm in the case of glutathione S-transferase activity,
1 Supported by USPHS Grant CA 21737, Bristol-Myers Squibb Com- nmol 2,6-dichborophenol-indophenol reduced/mm in the case of DT- pany Grant I00-R220, and Department of Defense Grant DAMD 17- diaphorase activity, and nmol p-nitrophenol formed/mm in the case of 94-J-4057. Descriptions of parts of this investigation have appeared in esterase activity]; PVDF, polyvinylidene difluoride. abstract form (L. Sreerama, M. Hedge, and N. E. Sladek. Proc. Am. 4 Monofunctional inducers are defined herein as agents that induce Assoc. Cancer Res., 35: 84, 1994). ALDH-3, glutathione S-transferases, and DT-diaphorase [NAD(P)H: 2 To whom requests for reprints should be addressed, at Department of quinone oxidoreductase; NQO1] but not cytochrome P450s IA1 and Pharmacology, University of Minnesota, 3-249 Millard Hall, 435 Del- 1A2; bifunctional inducers are defined herein as agents that induce all of aware Street SE., Minneapolis, MN 55455. these enzymes (Ref. 28 and references cited therein).
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proaches are that each is invasive, the latter two are labor Human stomach mucosa was provided by the Cooperative Hu- intensive, and the enzyme levels in serum are very bow on nib man Tissue Network (Midwest Division, Columbus, OH). Pu- (Ref. 15 and the present study). Another way of doing it would rifled stomach mucosa ALDH-3 and chicken antistomach mu- be to determine various phammacokinetic parameters, e.g., cosa ALDH-3 IgY were prepared as described previously (22). plasma half-life, of an appropriate test substrate before, during, Collection and further processing of saliva was essentially and after exposure to the suspected inducer (16); this approach as described by Takase et al. (23), except that hyaburonidase was is also invasive, not likeby to be as specific, and, in any event, not added to the collected saliva. Briefly, saliva samples (3-5 likely to be even more labor intensive. ml) were collected between 9 and 1 1 am and were placed into It occurred to us that if one or more of the relevant enzymes small beakers kept at 4#{176}C.DT1’ was added (final concentration is present in the saliva, it might be the ideal tissue/fluid to look 5 mM), and the samples were centrifuged at 9000 X g and 4#{176}C at. Thus, we searched the literature for reports documenting the for 15 mm. The supemnatant fractions thus obtained were used as presence of at least one of these enzymes in saliva. We were such when enzyme activities present therein were to be quanti- unable to find such a report. However, we did come across an fled. They were further processed when they were to be sub- investigation in which the presence of an aldehyde dehydroge- mitted to SDS-PAGE, immunoblot analysis, isoebectnic focus- ing, and/on when column chromatographic purification of the nase, apparently different from the known class 1, 2, and 3 abdehyde dehydnogenases and dubbed ALDH-V, in human sa- abdehyde dehydnogenase present therein was to be attempted. In those cases, they were desabted with the aid of PD-10 (Sephadex liva was demonstrated (17, 18). Although most of the reported G-25) columns (isoelectnic focusing of abdehyde dehydnoge- catalytic and physical properties of this enzyme did indeed nase) and/or concentrated in Centnicon-lO concentrators (Ami- appear to be different from those exhibited by ALDH-3, one, con Division, W. R. Grace & Co., Danvens, MA) by low-speed namely, isoebectnic point values, was not. Thus, we initiated centrifugation (isoelectnic focusing or column chromatographic experiments designed to further evaluate the identity of the purification of aldehyde dehydrogenase; SDS-PAGE and salivary aldehyde dehydnogenase and found it to indeed be immunobbot analysis of glutathione S-tnansfenases). Desalted ALDH-3. In the course of these investigations, we ascertained 9000 x g supemnatant fractions (obtained from 1.5 ml saliva) that, in addition to ALDH-3, glutathione S-tmansferases and were subjected to affinity chromatography on reactive blue DT-diaphonase were also present in the saliva and that levels of 2-Sepharose CL-6B as described previously (22) to eliminate each of these enzymes varied widely, but in approximately nonspecific proteins, and the resultant preparation was then direct proportion to each other. A review of the gender, age, concentrated as described above when SDS-PAGE/immunobbot mace, ethnicity, and dietary and other habits of the subjects who analysis for the presence of ALDH-3 was attempted. had contributed salivary samples strongly pointed to a direct Blood (15-20 ml) was collected in heparinized syringes and relationship between enzyme activities and coffee consumption. immediately centrifuged at 2000 X g and 4#{176}Cfor 15 mm. Rou- A subsequent review of the literature revealed that coffee con- tinely, the plasma thus obtained was then assayed for enzyme tains demonstrated inducens, e.g. , catechob, of these enzymes activities. In those cases where SDS-PAGE/immunobbot analysis ( 19). Further experimentation revealed that the continuous con- for the presence of ALDH-3 was to be performed, plasma samples sumption of relatively barge amounts of coffee did indeed result were first desabted and subjected to reactive blue 2-Sephanose in markedly increased salivary ALDH-3, glutathione S-trans- CL-6B affinity chromatography, after which the resultant fraction fenase, and DT-diaphorase levels as did the continuous con- of interest was concentrated, all as described above. sumption of relatively large quantities of broccoli, a vegetable The abdehyde dehydrogenase present in the saliva obtained known to (a) be rich in inducens of these enzymes (5, 6) and (b) from a single individual (NADPtdependent enzyme-catalyzed prevent experimental cancinogenesis (reviewed in Refs. 7, 9, and oxidation of benzabdehyde was 41 mIU/mb saliva) was purified by 20). successive DEAE-Sephaceb anion exchange chromatography, CM- Sephanose CL-6B cation exchange chromatography, and reactive MATERIALS AND METHODS blue 2-Sepharose CL-6B affinity chromatography as previously 4-Hydroperoxycycbophosphamide was supplied by Dr. J. described (22), except that elution of aldehyde dehydrogenase off Pohb (Asta Medica AG, Frankfurt, Germany). Purified human the affinity column was with 1, rather than 5, mst NAD .
glutathione S-transferases a, ji, and ‘ir and affinity-purified Ebectrotnansfer and immunobbot analysis of ALDH-3 and polycbonal antibodies specific for each of these isozymes (21) glutathione S-transfenases were essentially as described before were provided by Dr. A. J. Townsend (Department of Biochem- (2, 21, 22); antibody dilutions were 1:500 and 1:1000 in the case istry, Bowman Gray School of Medicine, Wake Forest Univer- of ALDH-3 and glutathione S-transferases, respectively. All other sity, Winston-Salem, NC). Antinabbit IgG alkaline phosphatase experimental procedures were as described previously (2, 22). conjugate was purchased from Sigma Chemical Co. (St. Louis, Double-reciprocal plots of initial rates versus substrate MO). Coffee was purchased from the University of Minnesota concentrations were used to estimate all Km values. Initial mates Food Services Cafeteria (University of Minnesota Medical were determined in duplicate for each of the six to eight sub- School, Minneapolis, MN). Broccoli was purchased from a local strate concentrations used to generate each value. Wilkinson market, washed, and cooked before consumption. All other weighted linear regression analysis (24) was used to fit lines to chemicals, reagents, and supplies were purchased from commen- the double-reciprocal plot values. cial sources or prepared as described previously (2, 22). Computer-assisted unweighted regression analysis was car- Human saliva and blood were obtained from healthy adult ned out using the STATView (Brainpower, Inc., Calabas, CA) male and female volunteers ranging from 20 to 55 years of age. statistical program to generate all other linear functions.
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Table I Aldehyde dehydro genase purified fro m human saliva” 123 Stomach Measurement Saliva Mucosa”
Specific activity 31,000 33,000 (mIU/mg protein) 7.35 !1 #{231} Yield (%) 51 60 Fold purification 1,340 423 6.85
“ Enzyme purification was as described in ‘‘ Materials and Meth- ods.’ ‘ Benzaldehyde (4 mM) and NAD (1 mM) were used as substrate 6.55 and cofactor, respectively, to monitor aldehyde dehydrogenase activity. #{149}1 Aldehyde dehydrogenase activity in the starting material (9000 X g :1 supernatant fraction of saliva) was 41 mIU/ml. :1 F, Values for purified stomach mucosa ALDH-3 are from a previous 5.85 publication (22); they are included for comparative purposes.
RESULTS 5.20 Preliminary studies revealed the presence of an NA(D)Pt 4.55 dependent enzyme that catalyzed the oxidation of benzaldehyde to benzoic acid in human saliva. The level (concentration) of this enzyme activity in the saliva of any given individual was 3.50 independent of the time of day at which the sample was cob- lected, on whether the sample was collected just before (10-15 mm), just after (10-15 mm), on long after (15-16 h) eating. Fig. I Isoelectric focusing of the aldehyde dehydrogenase purified However, it did decrease when the sample volume exceeded from human saliva. Purification and isoelectric focusing of the aldehyde approximately 10 ml. Isoebectnic focusing of the proteins present dehydrogenase present in human saliva were as described in “Materials in either 9000 x g on 105,000 X g supernatant fractions ob- and Methods.” Subjected to isoelectric focusing were isoelectnic point standards (Lane /), and amounts of purified stomach mucosa ALDH-3 tamed from saliva followed by staining for aldehyde dehydno- (Lane 2) and the aldehyde dehydrogenase purified from human saliva genase activity (benzabdehyde, octanal, and acetabdehyde as (Lane 3) sufficient to generate approximately 10-15 nmol NADH/min substrates and NAD as cofactor) suggested the presence of a (as determined by spectrophotometric assay) when benzaldehyde (4 single aldehyde dehydnogenase, one that exhibited an electro- mM) and NAD (I mM) were used as substrate and cofactor, respec- phonetic mobility similar, if not identical, to that exhibited by tively. Lane /, stained with Coomassie brilliant blue R-250 for the presence of proteins. Lanes 2 and 3, stained for aldehyde dehydrogenase stomach mucosa ALDH-3 (data not shown). Thus, the salivary activity as described in “Materials and Methods”; benzaldehyde (4 mM) enzyme was purified and a direct comparison of its physical and and NAD (4 mM) were used as the substrate and cofactor, respectively. kinetic properties with those of purified stomach mucosa ALDH-3 was made. The apparently pure salivary abdehyde dehydrogenase ex- hibited a specific activity of 31,000 mIU/mg protein (Table I), Like the stomach mucosa ALDH-3, the salivary ALDH-3 and isoebectnic point values characteristic of ALDH-3 (reviewed only poorly catalyzed the oxidation of abdophosphamide to in Refs. 22 and 25), although they were not exactly identical to carboxyphosphamide (Table 3). This characteristic distin- those exhibited by stomach mucosa ALDH-3 (Fig. 1). While guishes the ALDH-3 present in stomach mucosa and saliva from always fabling in the p1 mange of 5.7-6.4, the exact banding the ALDH-3s that are present in the two human tumor celb lines pattern of ALDH-3 seems to inexplicably vary somewhat with that have been, thus fan, booked at, namely, MCF-7 breast the tissue of origin (reviewed in Refs. 22 and 25). As judged by adenocarcinoma and colon C carcinoma, in that those in the both nondenatuming linear gradient PAGE and gel permeation latter catalyze the reaction at measurably greaten rates (2, 3, 22, chromatography on Sephacryl 5-200, its relative native molec- 26-28). ular weight was I 10,000 (data not shown). Antistomach mucosa Among individuals, salivary ALDH-3 bevels varied widely ALDH-3 IgY recognized the native (data not shown) as well as (Fig. 3). Lange intenindividual variations in salivary glutathione the denatured (Fig. 2) enzyme, and the relative subunit molec- S-transfenase and DT-diaphorase levels were also observed (Fig. ular weight was 54,500. As judged by Km values, the salivary 3). Indicative of some sort of coordinated regulation of the aldehyde dehydmogenase much preferred benzabdehyde to acet- expression/secretion of these enzymes was the fact that the aldehyde as a substrate, and NAD to NADP as a cofacton relative amounts of each in any given saliva sample appeared to (Table 2). It was only partially inhibited (<30%) by a high be directly rebated (Fig. 4). concentration of disulfiram (50 jiM) and was heat labile, i.e., Intenindividuab variability in the salivary levels of these catalytic activity was completely lost in less than 10 mm when enzymes could not be attributed to gender, race, ethnicity, it was incubated at 56#{176}C(data not presented). Like all known alcohol consumption, the use of tobacco, or the consumption of aldehyde dehydnogenases, the purified salivary enzyme also animal products. However, it did appear to be rebated to coffee exhibited estenolytic activity (8390 mIU/mg protein). Each of consumption since the salivary bevels of all three enzymes were, these physical and catalytic characteristics are essentially iden- on average, substantially higher in individuals who regularly ticab to those exhibited by stomach mucosa ALDH-3 (22). consumed this beverage (at least 150 ml daily) when compared
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A B
12
S .:wr 97.4 66.0 54.5 kDa -. 45.0 I ’
31.0
21.5 14.3
Fig. 2 Subunit molecular weight of the aldehyde dehydrogenase purified from human saliva as determined by SDS-PAGE and recognition of the denatured enzyme by antistomach mucosa ALDH-3 IgY. Purification and SDS-PAGE of the aldehyde dehydrogenase present in human saliva were as described in “Materials and Methods.” A, subjected to SDS-PAGE were molecular weight markers (Lane 1) and 3 jig each of purified stomach mucosa ALDH-3 (Lane 2) and the aldehyde dehydrogenase purified from human saliva (Lane 3). Molecular weight markers were lysozyme (14.3 kDa), trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), BSA monomer (66 kDa), and phosphorylase b (97.4 kDa). Proteins in each lane were visualized by staining with Coomassie brilliant blue R-250. A plot of log Mr versus mobility was used to estimate subunit molecular weights. B, purified stomach mucosa ALDH-3 and the aldehyde dehydrogenase purified from human saliva (3 jig each) were submitted to SDS-PAGE and then electrotransferred onto an Immobibon-PVDF transfer membrane. The membrane was then probed with antistomach mucosa ALDH-3 IgY as described in “Materials and Methods” to visualize stomach mucosa ALDH-3 (Lane I) and the aldehyde dehydnogenase purified from human saliva (Lane 2).
Table 2 Substrate and cofactor preferences of the aldehyde Table 3 Catalysis of aldophosphamide and benzabdehyde oxidation dehydrogenase purified from human saliva by the aldehyde dehydrogenase purified from human saliva: relative rates” Km ( i.M) nmol Aldophosphamide oxidized/mm/mg (1000) Stomach Source nmol Benzaldehyde oxidized/mm/mg Aldehyde (mM) Cofactor (mM) Saliva” Muscosa” Saliva 0.32 NAD (1) 465 505 Benzaldehyde (0.05-4) Stomach mucosa” 0.29 463 486 NADP (4) a Aldehyde dehydrogenase activity was quantified as described in NAD (1) 85,000 80,000 Acetaldehyde (25-200) “Materials and Methods”; aldophosphamide (160 i.M) or benzaldehyde NADP (4) 85,000 81,000 NAD (0.01-1) 40 54 (4 mM) was the substrate, and NAD (1 mM) was the cofactor. Benzaldehyde (4) NADP (0.1-4) 1,250 1,000 b The value for purified stomach mucosa ALDH-3 is from a pre- vious publication (27); it is included here for comparative purposes. “ Each value is the mean of three determinations. I’ Values for purified stomach mucosa ALDH-3 are from a previous publication (22); they are included here for comparative purposes. salivary ALDH-3, glutathione S-tmansfenase, and DT-diaphorase activities that we observed in individuals who consumed mela- tiveby large amounts of coffee on a daily basis was, in fact, to the average salivary bevels of these enzymes in individuals brought about by an agent(s) present in the coffee, most prob- who did not drink coffee at all (Figs. 3 and 4). ably largely catechob and/or hydroquinone, that coordinately Immunobbot analysis (Fig. 5) confirmed that the level of induced the expression/secretion of these enzymes. ALDH-3 was indeed relatively elevated in the saliva of individ- To further test this notion, salivary ALDH-3, glutathione uabs who regularly drank relatively barge amounts of coffee, and S-tnansferase, and DT-diaphonase activities were quantified in a revealed that salivary levels of glutathione S-tmansfemases a, ji, volunteer who alternately went without drinking coffee for and ‘n were each relatively elevated in such individuals. several days and drinking relatively large amounts of the bev- Roasted coffee beans are a rich source of catechob and erage for several days. The results of the experiment fully
other known inducens, e.g. , hydroquinone, of these enzymes supported the notion (Fig. 6). Again, immunobbot analysis (19). Thus, it seemed likely that the relatively elevated levels of (Fig. 7) confirmed that the bevels of ALDH-3 were relatively
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. A B
. S E 80 . S E 60 .. E 60 E 40 >. I- E E >
S I.- S 40 S I- > > 4 I- I- S 0 I- S S 0 (I) $ S C, 20 LU 0 LU S S S S S S N S I o N z S S z S I I I r=0.88 LU 30 a’ LU 20 S S a’ 10 20 30 40 a’ 0 a, 0 S S 0 ALDH-3 ACTIVITY, mIU/mI a’ I 8 - a’ 0 S a’ S S 0 05 $ §xaS S S 0 a’ 0 0 005 E =- 005
0 E ALDH-3 GST DT-D ALDH-3 GST DT-D 80 >- I- Fig. 3 ALDH-3, glutathione S-transferase, and DT-diaphorase levels > in human saliva. Collection and processing of saliva and determinations I- 60 U of protein concentrations and enzyme activities were as described in 4 “Materials and Methods.” Benzaldehyde (4 mM) and NADP (4 mM) LU were used as substrate and cofactor, respectively, to quantify ALDH-3 C” 40 4 activity. Reduced glutathione (5 mM) and 1-chloro-2,4-dinitrobenzene (I mM) were used to quantify glutathione S-transferase (GS7) activity. 0 p.M), jiM), 2,6-Dichlorophenol-indophenol (40 NADH (160 and dicum- 0. 20 arol (10 jiM) were used as substrate, cofactor, and inhibitor, respec- 4 tively, to quantify DT-diaphorase (DT-D) activity. Points are the mean l. of duplicate determinations made on single samples (3-5 ml) collected from each donor. Enzyme activity was normalized for salivary volume 10 20 30 40 (A) and salivary protein (B). Overall mean ± SD values were 9 ± 9 mIU/ml saliva and 5 ± 5 mIU/mg salivary 9000 X g supernatant protein ALDH-3 ACTIVITY, mIU/mI for ALDH-3, 36 ± 21 mIU/ml saliva and 20 ± 12 mIU/mg salivary 9000 X g supernatant protein for glutathione S-transferase, and 21 ± 19 mIU/ml saliva and 1 1 ± 11 mIU/mg salivary 9000 X g supernatant E protein for DT-diaphorase. #{149},subjects who consumed at least 150 ml of coffee daily; mean ± SD values were 15 ± 12 mlU/ml saliva and 8 ± E 6 mIU/mg salivary 9000 X g supernatant protein for ALDH-3, 51 ± 21 80 mIU/ml saliva and 28 ± 12 mIU/mg salivary 9000 X g supernatant >- I- protein for glutathione S-transferase, and 31 ± 22 mIU/ml saliva and > 17 ± 12 mIU/mg salivary 9000 X g supernatant protein for DT- I- 60 diaphorase. 0, subjects who did not drink coffee at all; mean ± SD U 4 values were 5 ± 2 mIU/ml saliva and 3 ± 1 mIU/mg salivary 9000 X LU g supernatant protein for ALDH-3, 23 ± 7 mIU/ml saliva and 12 ± 3 C” 40 mIU/mg salivary 9000 X g supernatant protein for glutathione S- 4 transferase, and 1 1 ± 8 mIU/ml saliva and 6 ± 4 mIU/mg salivary 900() 0 x g supernatant protein for DT-diaphorase. In all cases, salivary enzyme a. 20 activities of coffee-drinking volunteers were significantly (P 0.005) 4 greater than were those of volunteers who did not drink coffee. I- elevated in salivary samples taken during the periods of high 20 40 60 80 coffee consumption, and revealed that salivary bevels of gluta- GST ACTIVITY, mIU/mI thione S-tnansfenases a, ji, and ‘rr were each relatively elevated during these periods. Fig. 4 ALDH-3, glutathione S-transferase (GST), and DT-diaphorase Numerous vegetables, e.g., crucifers such as broccoli, are levels in human saliva. In the investigation presented in Fig. 3, one or known to coordinately induce glutathione S-transferase and DT- more of the three enzyme activities of interest were quantified in single samples obtained from each of 33 subjects; all three were quantified in diaphonase activities in various models (5, 6). Furthermore, 25 subjects. Correlations between the magnitudes of enzyme activities serum levels of glutathione S-transferase a were elevated in in the latter are shown here. #{149},subjects who consumed at least 150 ml subjects who had consumed relatively barge amounts of brussels of coffee daily. 0, subjects who did not drink coffee at all.
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