J Cancer Res Clin Oncol (1990) 116:379-383
C~ilcer ~esearch Clinical 9 9 Springer-Verlag 1990
Detoxifying enzymes in human ovarian tissues: comparison of normal and tumor tissues and effects of chemotherapy * Zora Djuric 1,2, Vinay K. Malviya 1, Gunter Deppe 1, John M. Malone Jr l, Dawn L. McGunagle 1, Lance K. Heilbrun 2, Bruce A. Reading 2, and William D. Lawrence 3 1 Departments of t Obstetrics and Gynecology, 2Internal Medicine and 3Pathology, Wayne State University, Detroit, MI 48201, USA Received 27 December 1989/Accepted 27 April 1990
Summary. M a n y a n t i c a n c e r d r u g s exert their c y t o t o x i c effects via f o r m a t i o n o f o x y g e n free radicals. C e l l u l a r thiols, g l u t a t h i o n e ( G S H ) - d e p e n d e n t e n z y m e s a n d o t h e r r e d o x enzymes are i n v o l v e d in the m e t a b o l i s m o f these a n t i c a n c e r d r u g s a n d o f the o x y g e n free r a d i c a l s t h a t m a y be g e n e r a t e d d u r i n g their m e t a b o l i s m . W e q u a n t i f i e d these b i o c h e m i c a l p a r a m e t e r s in c y t o s o l f r o m h u m a n o v a r i a n tissues. W e c o m p a r e d n o n - p r o t e i n thiol levels, G S H transferase, G S H p e r o x i d a s e , s u p e r o x i d e d i s m u tase, catalase, D T d i a p h o r a s e a n d a l d e h y d e d e h y d r o g e nase activity in serous o v a r i a n t u m o r s (n = 15), o t h e r m a l i g n a n t o v a r i a n t u m o r s ( n = 1 2 ) , b e n i g n o v a r i a n tissue (n = 10) a n d h i s t o l o g i c a l l y n o r m a l o v a r i a n tissue (n = 12). M e a n G S H t r a n s f e r a s e a n d D T d i a p h o r a s e activities were similar in serous a n d o t h e r m a l i g n a n t o v a r i a n tumors. G S H transferase activity was d e c r e a s e d in m a l i g n a n t tissues relative to n o r m a l a n d b e n i g n tissues. M e a n D T d i a p h o r a s e a n d s u p e r o x i d e d i s m u t a s e activities were increased in the m a l i g n a n t tissues, a l t h o u g h this was n o t statistically significant. T h e m e a n levels o f all a n z y m e s except s u p e r o x i d e d i s m u t a s e a n d a l d e h y d e d e h y d r o g e nase in b e n i g n tissues were fairly similar to the m e a n levels f o u n d in n o r m a l tissue samples. Tissues f r o m p a tients w i t h serous o v a r i a n t u m o r s , w h o h a d received c y c l o p h o s p h a m i d e a n d cisplatin p r i o r to surgery, also were a n a l y z e d (n = 7). E x c e p t for a l d e h y d e d e h y d r o g e nase, all the p a r a m e t e r s m e a s u r e d were d e c r e a s e d in these s a m p l e s relative to serous tissue f r o m u n t r e a t e d patients. These b i o c h e m i c a l a n a l y s e s m a y be useful in u n d e r s t a n d ing the m e c h a n i s m s i n v o l v e d in the r e s p o n s e to c h e m o therapy.
Key words: O v a r i a n c a n c e r - C h e m o t h e r a p y - G l u t a thione - Redox enzymes * Partial support for this study was obtained from the Comprehensive Cancer Center of Metropolitan Detroit, core grant CA-22453, National Institutes of Health Abbreviations: ANOVA, analysis of variance; GSH, glutathione Offprint requests to: Z. Djuric, Division of Hematology and Oncology, Department of Internal Medicine, Wayne State University, P.O. Box 02188, Detroit, MI 48202, USA
Introduction O v a r i a n c a n c e r is the l e a d i n g cause o f g y n e c o l o g i c a l cancer d e a t h in the U n i t e d States ( B r e n n e r a n d G r e c o 1985). C o m b i n a t i o n c h e m o t h e r a p y is the m a i n s t a y o f p o s t o p e r a t i v e a d j u v a n t t r e a t m e n t for o v a r i a n c a n c e r a n d results in a n a v e r a g e c o m p l e t e r e s p o n s e r a t e o f 4 0 % (Brenner a n d G r e c o 1985). T h e efficacy o f these c h e m o t h e r a p e u t i c r e g i m e n s m a y be limited b y the a b i l i t y o f the t u m o r tissue to m e t a b o l i z e the a d m i n i s t e r e d drug(s). W e have e x a m i n e d several d e t o x i f i c a t i o n systems in h u m a n o v a r i a n tissues t h a t m a y be i n v o l v e d in the m e t a b o l i s m o f c h e m o t h e r a p e u t i c agents t h e r e b y affecting the r e s p o n s e to c h e m o t h e r a p y .
Materials and methods Tissue samples. Ovarian tissue was removed during surgery and immediately placed in ice-cold saline. The tissue was sliced and sections for biochemical analyses were selcted by the pathologists. Adjacent sections of the analyzed tissue were used for histological diagnosis. The tissue was kept in ice-cold saline until it was homogenized in four volumes of 250 mM sucrose, 10 mM TRIS, pH 7.4, and 1 mM EDTA. Cytosol was prepared by centrifugation of the homogenate at 105 000 g for 1 h. Chemotherapy. All patients in the study designated as "prior chemotherapy" were treated 1-3 months prior to surgery with multiple courses of therapy containing platinum and cyclophosphamide. The exact chemotherapy each patient received is listed: (a) 9 courses of 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan, last course 2 months prior to surgery; (b) 7 courses of 50 mg/m 2 Adriamycin, 50 mg/m z cisplatin and 500 mg/m 2 cytoxan, last course 2 months prior to surgery; (c) 6 courses of 100 mg/m 2 cisplatin and 600 mg/m 2 cytoxan followed by 6 courses of 50 mg/m 2 cisplatin, last course 3 months prior to surgery; (d) 5 courses of 50 mg/m z carboplatin and 500 mg/ m 2 cytoxan, last course 2 months prior to surgery; (e) 6 courses of 50 mg/m z Adriamycin, 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan, last course t month prior to surgery; (f) 9 courses of 50 mg/m 2 Adriamycin, 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan followed by 6 courses of 100 mg/m z cisplatin and 100 mg/m 2 VP16, last course 1 month prior to surgery; (g) 8 courses of 50 mg/m 2 Adriamycin, 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan, subsequent 14 courses of 50 mg/m 2 cisplatin and finally 5 courses of 50 mg/m z cis-
380 platin, last course 1 month prior to surgery; (h) 6 courses of 100 mg/ m 2 cisplatin and 600 mg/m 2 cytoxan, subsequent 2 courses of 300 mg/m2 cis-dichloro-trans-hydroxy,bis-iso-propylamine platinum IV and finally 4 courses of 50 mg/m2 Adriamycin, 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan, last course 3 months prior to surgery.
Enzyme assays. All chemicals and biochemicals were obtained from Sigma Chemical Co. (St. Louis, Mo) in the purest grade available. Enzyme assays described below were performed at 25" C in a Hewlett-Packard HP8451 spectrophotometer. Each assay was performed in duplicate, at the minimum, for each sample. Every enzyme could not be assayed in some samples because of limitations in tissue size. Glutathione (GSH) peroxidase acitivity was determined in a coupled assay (Floh6 and Gfinzler 1984) by measuring the H2Oz-dependent oxidation of NADPH at 340 nm (e = 6.22 mM %m- 1). Incubations were conducted with 1 mg/ml cytosolic protein, 100 mM potassium phosphate, pH 7.4, I mM EDTA, 1 mM sodium azide, 10 mM GSH, 0.15 mM NADPH, 0.24 unit/ml glutathione reductase and 0.15 mM H202. Background rates of NADPH oxidation were determined prior to addition to H202. Superoxide dismutase activity was determined from the ability of cytosol to inhibit the xanthine-oxidase-dependent rate of cytochrome c reduction (Floh6 and (3tting 1984). Incubations contained 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, 10 gM sodium azide, 30 gM succinoylated cytochrome c, 1 unit/ml xanthine oxidase, 500 gM hypoxanthine and varying amounts of cytosol (0.01-1 mg protein/ml). Reactions were followed at 550 nm (e = 21 mM- lcm 1) and one unit of activity was defined as the amount of cytosol required to inhibit the rate of succinoylated cytochrome c reduction by 50%. Non-protein thiols in cytosol were measured by the colorimetric reaction with Ellman's reagent (Ellman 1959). Briefly, an equal volume of 10% trichloroacetic acid was mixed with cytosol or standard GSH solution. After centrifugation for 1 rain at 13 000 g, 25 gl superhatant was added to 700 gl 100 gM 5,5'-dithiobis(2-nitrobenzoic acid) in 0.5% sodium bicarbonate. After 10 min at room temperature, the absorbance of the samples at 412 nm was measured. Catalase activity was measured by the cytosol-catalyzed rate of H202 degradation at 240 nm (e = 3.94 M - 1 cm 1) (Aebi 1984). Assays were conducted with 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, 56 mM H202 and 0.3 mg/mt cytosol. GSH transferase activity was determined from the rate of lchloro-2,4-dinitrobenzene conjugation with GSH (Habig et al. 1974). Assays consisted of 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, 5 mM GSH, 1 mM l-chloro-2,4-dinitrobenzeneand 0.1 mg/ml cytosolic protein. Rates were followed at 340 nm (e = 9.6 mM lcm- 1). DT diaphorase activity was determined from the rate of cytochrome c reduction in cytosolic incubations with NADH and menadione (Lind and Ernster 1974). Incubations contained 1 mg/ml cytosolic protein, 30 gM succinoylated cytochrome c, 50 gM menadione, 1 mM NADH and 100 mM potassium phosphate, pH 7.4. Some incubations also contained 0.1 mg/ml superoxide dismutase and the rate not inhibited by superoxide dismutase was used as a measure of DT diaphorase. The rate of cytochrome c reduction was followed at 550 nm (e=21 m M - l c m i). Aldehyde dehydrogenase activity was measured from the rate of benzaldehyde-dependent NAD reduction (Domeyer and Sladek 1980), which was followed at 340 nm (e = 6.22 mM lcm- 1). Assays were conducted with 32 mM sodium pyrophosphate, pH 7.4, 4 mM benzaldehyde, 200 mM pyrazole, 4 mM NAD and 1 mg/ml cytosolic protein.
Statistical methods. The enzyme levels were analyzed as seven dependet variables to determine the effect of ovarian tissue type (normal, benign, serous, other malignant) on the mean enzyme level. This was performed with one-way, unbalanced, fixed effects analysis of variance (ANOVA) methods using a general linear-models approach (Freund and Littell 1986). Separate univariate ANOVA models were fit for each of the seven enzymes. Tests for normality
of the dependent variable were performed before proceeding with the ANOVA. A rank transformation of four of the enzymes (GSH transferase, superoxide dismutase, DT diaphorase and aldehyde dehydrogenase) was necessary to satisfy the ANOVA assumption of normality. Analyses were carried out on the rank-transformed values of those four enzymes, and on the raw values of the remaining three enzymes. However, for presentation purposes, the arithmetic means of the untransformed data are reported as summary statistics (Conover and Iman 1981). Differences between serous tumor tissues by prior chemotherapy status (yes/no) were evaluated by two-sample t-tests. All tests were two-tailed. When investigating the significant main effects of tissue type found in the ANOVA (i.e., pairwise comparisons of tissue types), the Tukey multiple comparisons procedure was used to maintain the experimentwise type-I error rate at 0.05 (Freund and Littell 1986). For each of the various tissue groups compared, there were 6-15 observations. The data were viewed as resulting from seven separate (1 x 4) complete one-way-layout experimental designs.
Results Tissue samples. O v a r i a n tissue was o b t a i n e d from postm e n o p a u s a l w o m e n . Tissues were placed in ice-cold saline i m m e d i a t e l y after excision a n d kept o n ice until cytosol could be prepared, typically w i t h i n 2 h. I n v e s t i g a t i o n of v a r i o u s storage techniques indicated that enzymatic activity was preserved for at least 1 day if whole tissue was kept in ice-cold saline prior to p r e p a r a t i o n of cytosol. Freezing of either the tissue or the cytosol substantially reduced enzymatic activity. Histologically n o r m a l o v a r i a n tissue was o b t a i n e d from 12 patients with cancer of the cervix or e n d o m e trium; m e a n (+_ s t a n d a r d error) p a t i e n t age was 58 +_ 11 years (range 44-72). B e n i g n o v a r i a n tissue was o b t a i n e d from 10 patients with hyperplasia, a d e n o m a s or adenofib r o m a s , a n d their m e a n age was 48 + 12 years (range 2 5 64). T h e m a j o r i t y o f m a l i g n a n t o v a r i a n t u m o r s analyzed were o f the serous type. U n t r e a t e d serous o v a r i a n t u m o r tissue was o b t a i n e d f r o m 15 patients in clinical stage III or IV, with a m e a n age of 62 __ 9 years (range 40-80). A d d i t i o n a l m a l i g n a n t t u m o r tissue was o b t a i n e d from 12 end o m e t r o i d , g r a n u l o s a cell or m u c i n o u s o v a r i a n a d e n o c a r c i n o m a s . M e a n p a t i e n t age was 62 _+ 14 years (range 39 82), a n d all b u t 2 patients were p o s t m e n o p a u s a l , T r e a t e d serous o v a r i a n tissue also was o b t a i n e d from 8 patients in clinical stage III or IV, with a m e a n age o f 57 + 4 years (range 50-64). Enzyme levels. E n z y m e levels in histologically n o r m a l o v a r i a n tissues, b e n i g n tissue, serous t u m o r s a n d other m a l i g n a n t t u m o r tissues are s h o w n in T a b l e 1. Patients w h o h a d received c h e m o t h e r a p y p r i o r to surgery were excluded in the c o m p a r i s o n of n o r m a l , b e n i g n a n d malign a n t tissues. The detoxifying enzyme, G S H transferase, differed significantly by tissue type ( P = 0 . 0 0 2 ) . T u k e y m u l t i p l e c o m p a r i s o n s revealed t h a t the m e a n G S H transferase level o f " o t h e r m a l i g n a n t t u m o r " tissue was significantly lower t h a n the m e a n for either n o r m a l or b e n i g n tissue ( P < 0.05 in each case). T h e o n l y other enzyme to differ significantly b y tissue type was G S H peroxidase ( P = 0,037). O n l y one pair o f m e a n s was responsible for this: serous versus other m a l i g n a n t t u m o r s ( P < 0 . 0 5 by
381 Table 1. Mean cytosolic enzyme levels in human ovarian tissues from 49 patients without prior chemotherapy Parameter a
Non-proteinthiols GSH transferase GSH peroxidase Catalase Superoxide dismutase DT diaphorase Aldehyde dehydrogenase
Level in ovarian tissue b
P value c
Normal
Benign
228 • 53 (10) 609 • d (9) 33.8 • 4.4 (11) 36.6 • 10.2 (10) 5.1 • 1.8 (10) 1.87• 0.55 (12) 2.42• 1.67 (9)
217 • 475 _+ 26.7 • 37.4 • 6.0 _+ 1.49• 1.41•
64 60 a 5.0 6 2.6 0.27 0.62
(8) (8) (10) (9) (9) (10) (8)
Serous
Other malignant
379 • 67 (11) 304 • 38 (14) 40.5 • 3.6 e (15) 38.3 • 7.6 (8) 7.4 • 1.6 (15) 3.35• 0.74 (15) 0.37• 0.21 (8)
269 • 70 (9) 277 • 98 d (11) 24.0 • 4.9 e (10) 22.7 • 10.2 (7) 9.1 • 1.9 (11) 3.05• 1.14 (12) 1.48• 0.52 (9)
0,255 0.002 0.037 0.616 0.232 0.217 0.273
" Assay procedures are described in Materials and methods. Values are expressed in nmol min 1mg cytosolic protein 1 except for catalase (gmol m i n i mg- 1), superoxide dismutase (units/nag) and non-protein thiols (nmol/ml cytosol) b Values given are the mean _+ the standard error, and the number of samples (n). The majority of malignant ovarian tissue was of the serous type The statistical significance of the difference in means across the four tissue types was determined using one-way unbalanced fixedeffects analysis of variance d Means for normal and benign tissue are significantly different (P < 0.05 by the Tukey multiple comparisons method) from other malignant tissue e Mean for serous tissue is significantly different (P
C~ilcer ~esearch Clinical 9 9 Springer-Verlag 1990
Detoxifying enzymes in human ovarian tissues: comparison of normal and tumor tissues and effects of chemotherapy * Zora Djuric 1,2, Vinay K. Malviya 1, Gunter Deppe 1, John M. Malone Jr l, Dawn L. McGunagle 1, Lance K. Heilbrun 2, Bruce A. Reading 2, and William D. Lawrence 3 1 Departments of t Obstetrics and Gynecology, 2Internal Medicine and 3Pathology, Wayne State University, Detroit, MI 48201, USA Received 27 December 1989/Accepted 27 April 1990
Summary. M a n y a n t i c a n c e r d r u g s exert their c y t o t o x i c effects via f o r m a t i o n o f o x y g e n free radicals. C e l l u l a r thiols, g l u t a t h i o n e ( G S H ) - d e p e n d e n t e n z y m e s a n d o t h e r r e d o x enzymes are i n v o l v e d in the m e t a b o l i s m o f these a n t i c a n c e r d r u g s a n d o f the o x y g e n free r a d i c a l s t h a t m a y be g e n e r a t e d d u r i n g their m e t a b o l i s m . W e q u a n t i f i e d these b i o c h e m i c a l p a r a m e t e r s in c y t o s o l f r o m h u m a n o v a r i a n tissues. W e c o m p a r e d n o n - p r o t e i n thiol levels, G S H transferase, G S H p e r o x i d a s e , s u p e r o x i d e d i s m u tase, catalase, D T d i a p h o r a s e a n d a l d e h y d e d e h y d r o g e nase activity in serous o v a r i a n t u m o r s (n = 15), o t h e r m a l i g n a n t o v a r i a n t u m o r s ( n = 1 2 ) , b e n i g n o v a r i a n tissue (n = 10) a n d h i s t o l o g i c a l l y n o r m a l o v a r i a n tissue (n = 12). M e a n G S H t r a n s f e r a s e a n d D T d i a p h o r a s e activities were similar in serous a n d o t h e r m a l i g n a n t o v a r i a n tumors. G S H transferase activity was d e c r e a s e d in m a l i g n a n t tissues relative to n o r m a l a n d b e n i g n tissues. M e a n D T d i a p h o r a s e a n d s u p e r o x i d e d i s m u t a s e activities were increased in the m a l i g n a n t tissues, a l t h o u g h this was n o t statistically significant. T h e m e a n levels o f all a n z y m e s except s u p e r o x i d e d i s m u t a s e a n d a l d e h y d e d e h y d r o g e nase in b e n i g n tissues were fairly similar to the m e a n levels f o u n d in n o r m a l tissue samples. Tissues f r o m p a tients w i t h serous o v a r i a n t u m o r s , w h o h a d received c y c l o p h o s p h a m i d e a n d cisplatin p r i o r to surgery, also were a n a l y z e d (n = 7). E x c e p t for a l d e h y d e d e h y d r o g e nase, all the p a r a m e t e r s m e a s u r e d were d e c r e a s e d in these s a m p l e s relative to serous tissue f r o m u n t r e a t e d patients. These b i o c h e m i c a l a n a l y s e s m a y be useful in u n d e r s t a n d ing the m e c h a n i s m s i n v o l v e d in the r e s p o n s e to c h e m o therapy.
Key words: O v a r i a n c a n c e r - C h e m o t h e r a p y - G l u t a thione - Redox enzymes * Partial support for this study was obtained from the Comprehensive Cancer Center of Metropolitan Detroit, core grant CA-22453, National Institutes of Health Abbreviations: ANOVA, analysis of variance; GSH, glutathione Offprint requests to: Z. Djuric, Division of Hematology and Oncology, Department of Internal Medicine, Wayne State University, P.O. Box 02188, Detroit, MI 48202, USA
Introduction O v a r i a n c a n c e r is the l e a d i n g cause o f g y n e c o l o g i c a l cancer d e a t h in the U n i t e d States ( B r e n n e r a n d G r e c o 1985). C o m b i n a t i o n c h e m o t h e r a p y is the m a i n s t a y o f p o s t o p e r a t i v e a d j u v a n t t r e a t m e n t for o v a r i a n c a n c e r a n d results in a n a v e r a g e c o m p l e t e r e s p o n s e r a t e o f 4 0 % (Brenner a n d G r e c o 1985). T h e efficacy o f these c h e m o t h e r a p e u t i c r e g i m e n s m a y be limited b y the a b i l i t y o f the t u m o r tissue to m e t a b o l i z e the a d m i n i s t e r e d drug(s). W e have e x a m i n e d several d e t o x i f i c a t i o n systems in h u m a n o v a r i a n tissues t h a t m a y be i n v o l v e d in the m e t a b o l i s m o f c h e m o t h e r a p e u t i c agents t h e r e b y affecting the r e s p o n s e to c h e m o t h e r a p y .
Materials and methods Tissue samples. Ovarian tissue was removed during surgery and immediately placed in ice-cold saline. The tissue was sliced and sections for biochemical analyses were selcted by the pathologists. Adjacent sections of the analyzed tissue were used for histological diagnosis. The tissue was kept in ice-cold saline until it was homogenized in four volumes of 250 mM sucrose, 10 mM TRIS, pH 7.4, and 1 mM EDTA. Cytosol was prepared by centrifugation of the homogenate at 105 000 g for 1 h. Chemotherapy. All patients in the study designated as "prior chemotherapy" were treated 1-3 months prior to surgery with multiple courses of therapy containing platinum and cyclophosphamide. The exact chemotherapy each patient received is listed: (a) 9 courses of 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan, last course 2 months prior to surgery; (b) 7 courses of 50 mg/m 2 Adriamycin, 50 mg/m z cisplatin and 500 mg/m 2 cytoxan, last course 2 months prior to surgery; (c) 6 courses of 100 mg/m 2 cisplatin and 600 mg/m 2 cytoxan followed by 6 courses of 50 mg/m 2 cisplatin, last course 3 months prior to surgery; (d) 5 courses of 50 mg/m z carboplatin and 500 mg/ m 2 cytoxan, last course 2 months prior to surgery; (e) 6 courses of 50 mg/m z Adriamycin, 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan, last course t month prior to surgery; (f) 9 courses of 50 mg/m 2 Adriamycin, 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan followed by 6 courses of 100 mg/m z cisplatin and 100 mg/m 2 VP16, last course 1 month prior to surgery; (g) 8 courses of 50 mg/m 2 Adriamycin, 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan, subsequent 14 courses of 50 mg/m 2 cisplatin and finally 5 courses of 50 mg/m z cis-
380 platin, last course 1 month prior to surgery; (h) 6 courses of 100 mg/ m 2 cisplatin and 600 mg/m 2 cytoxan, subsequent 2 courses of 300 mg/m2 cis-dichloro-trans-hydroxy,bis-iso-propylamine platinum IV and finally 4 courses of 50 mg/m2 Adriamycin, 50 mg/m 2 cisplatin and 500 mg/m 2 cytoxan, last course 3 months prior to surgery.
Enzyme assays. All chemicals and biochemicals were obtained from Sigma Chemical Co. (St. Louis, Mo) in the purest grade available. Enzyme assays described below were performed at 25" C in a Hewlett-Packard HP8451 spectrophotometer. Each assay was performed in duplicate, at the minimum, for each sample. Every enzyme could not be assayed in some samples because of limitations in tissue size. Glutathione (GSH) peroxidase acitivity was determined in a coupled assay (Floh6 and Gfinzler 1984) by measuring the H2Oz-dependent oxidation of NADPH at 340 nm (e = 6.22 mM %m- 1). Incubations were conducted with 1 mg/ml cytosolic protein, 100 mM potassium phosphate, pH 7.4, I mM EDTA, 1 mM sodium azide, 10 mM GSH, 0.15 mM NADPH, 0.24 unit/ml glutathione reductase and 0.15 mM H202. Background rates of NADPH oxidation were determined prior to addition to H202. Superoxide dismutase activity was determined from the ability of cytosol to inhibit the xanthine-oxidase-dependent rate of cytochrome c reduction (Floh6 and (3tting 1984). Incubations contained 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, 10 gM sodium azide, 30 gM succinoylated cytochrome c, 1 unit/ml xanthine oxidase, 500 gM hypoxanthine and varying amounts of cytosol (0.01-1 mg protein/ml). Reactions were followed at 550 nm (e = 21 mM- lcm 1) and one unit of activity was defined as the amount of cytosol required to inhibit the rate of succinoylated cytochrome c reduction by 50%. Non-protein thiols in cytosol were measured by the colorimetric reaction with Ellman's reagent (Ellman 1959). Briefly, an equal volume of 10% trichloroacetic acid was mixed with cytosol or standard GSH solution. After centrifugation for 1 rain at 13 000 g, 25 gl superhatant was added to 700 gl 100 gM 5,5'-dithiobis(2-nitrobenzoic acid) in 0.5% sodium bicarbonate. After 10 min at room temperature, the absorbance of the samples at 412 nm was measured. Catalase activity was measured by the cytosol-catalyzed rate of H202 degradation at 240 nm (e = 3.94 M - 1 cm 1) (Aebi 1984). Assays were conducted with 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, 56 mM H202 and 0.3 mg/mt cytosol. GSH transferase activity was determined from the rate of lchloro-2,4-dinitrobenzene conjugation with GSH (Habig et al. 1974). Assays consisted of 100 mM potassium phosphate, pH 7.4, 1 mM EDTA, 5 mM GSH, 1 mM l-chloro-2,4-dinitrobenzeneand 0.1 mg/ml cytosolic protein. Rates were followed at 340 nm (e = 9.6 mM lcm- 1). DT diaphorase activity was determined from the rate of cytochrome c reduction in cytosolic incubations with NADH and menadione (Lind and Ernster 1974). Incubations contained 1 mg/ml cytosolic protein, 30 gM succinoylated cytochrome c, 50 gM menadione, 1 mM NADH and 100 mM potassium phosphate, pH 7.4. Some incubations also contained 0.1 mg/ml superoxide dismutase and the rate not inhibited by superoxide dismutase was used as a measure of DT diaphorase. The rate of cytochrome c reduction was followed at 550 nm (e=21 m M - l c m i). Aldehyde dehydrogenase activity was measured from the rate of benzaldehyde-dependent NAD reduction (Domeyer and Sladek 1980), which was followed at 340 nm (e = 6.22 mM lcm- 1). Assays were conducted with 32 mM sodium pyrophosphate, pH 7.4, 4 mM benzaldehyde, 200 mM pyrazole, 4 mM NAD and 1 mg/ml cytosolic protein.
Statistical methods. The enzyme levels were analyzed as seven dependet variables to determine the effect of ovarian tissue type (normal, benign, serous, other malignant) on the mean enzyme level. This was performed with one-way, unbalanced, fixed effects analysis of variance (ANOVA) methods using a general linear-models approach (Freund and Littell 1986). Separate univariate ANOVA models were fit for each of the seven enzymes. Tests for normality
of the dependent variable were performed before proceeding with the ANOVA. A rank transformation of four of the enzymes (GSH transferase, superoxide dismutase, DT diaphorase and aldehyde dehydrogenase) was necessary to satisfy the ANOVA assumption of normality. Analyses were carried out on the rank-transformed values of those four enzymes, and on the raw values of the remaining three enzymes. However, for presentation purposes, the arithmetic means of the untransformed data are reported as summary statistics (Conover and Iman 1981). Differences between serous tumor tissues by prior chemotherapy status (yes/no) were evaluated by two-sample t-tests. All tests were two-tailed. When investigating the significant main effects of tissue type found in the ANOVA (i.e., pairwise comparisons of tissue types), the Tukey multiple comparisons procedure was used to maintain the experimentwise type-I error rate at 0.05 (Freund and Littell 1986). For each of the various tissue groups compared, there were 6-15 observations. The data were viewed as resulting from seven separate (1 x 4) complete one-way-layout experimental designs.
Results Tissue samples. O v a r i a n tissue was o b t a i n e d from postm e n o p a u s a l w o m e n . Tissues were placed in ice-cold saline i m m e d i a t e l y after excision a n d kept o n ice until cytosol could be prepared, typically w i t h i n 2 h. I n v e s t i g a t i o n of v a r i o u s storage techniques indicated that enzymatic activity was preserved for at least 1 day if whole tissue was kept in ice-cold saline prior to p r e p a r a t i o n of cytosol. Freezing of either the tissue or the cytosol substantially reduced enzymatic activity. Histologically n o r m a l o v a r i a n tissue was o b t a i n e d from 12 patients with cancer of the cervix or e n d o m e trium; m e a n (+_ s t a n d a r d error) p a t i e n t age was 58 +_ 11 years (range 44-72). B e n i g n o v a r i a n tissue was o b t a i n e d from 10 patients with hyperplasia, a d e n o m a s or adenofib r o m a s , a n d their m e a n age was 48 + 12 years (range 2 5 64). T h e m a j o r i t y o f m a l i g n a n t o v a r i a n t u m o r s analyzed were o f the serous type. U n t r e a t e d serous o v a r i a n t u m o r tissue was o b t a i n e d f r o m 15 patients in clinical stage III or IV, with a m e a n age of 62 __ 9 years (range 40-80). A d d i t i o n a l m a l i g n a n t t u m o r tissue was o b t a i n e d from 12 end o m e t r o i d , g r a n u l o s a cell or m u c i n o u s o v a r i a n a d e n o c a r c i n o m a s . M e a n p a t i e n t age was 62 _+ 14 years (range 39 82), a n d all b u t 2 patients were p o s t m e n o p a u s a l , T r e a t e d serous o v a r i a n tissue also was o b t a i n e d from 8 patients in clinical stage III or IV, with a m e a n age o f 57 + 4 years (range 50-64). Enzyme levels. E n z y m e levels in histologically n o r m a l o v a r i a n tissues, b e n i g n tissue, serous t u m o r s a n d other m a l i g n a n t t u m o r tissues are s h o w n in T a b l e 1. Patients w h o h a d received c h e m o t h e r a p y p r i o r to surgery were excluded in the c o m p a r i s o n of n o r m a l , b e n i g n a n d malign a n t tissues. The detoxifying enzyme, G S H transferase, differed significantly by tissue type ( P = 0 . 0 0 2 ) . T u k e y m u l t i p l e c o m p a r i s o n s revealed t h a t the m e a n G S H transferase level o f " o t h e r m a l i g n a n t t u m o r " tissue was significantly lower t h a n the m e a n for either n o r m a l or b e n i g n tissue ( P < 0.05 in each case). T h e o n l y other enzyme to differ significantly b y tissue type was G S H peroxidase ( P = 0,037). O n l y one pair o f m e a n s was responsible for this: serous versus other m a l i g n a n t t u m o r s ( P < 0 . 0 5 by
381 Table 1. Mean cytosolic enzyme levels in human ovarian tissues from 49 patients without prior chemotherapy Parameter a
Non-proteinthiols GSH transferase GSH peroxidase Catalase Superoxide dismutase DT diaphorase Aldehyde dehydrogenase
Level in ovarian tissue b
P value c
Normal
Benign
228 • 53 (10) 609 • d (9) 33.8 • 4.4 (11) 36.6 • 10.2 (10) 5.1 • 1.8 (10) 1.87• 0.55 (12) 2.42• 1.67 (9)
217 • 475 _+ 26.7 • 37.4 • 6.0 _+ 1.49• 1.41•
64 60 a 5.0 6 2.6 0.27 0.62
(8) (8) (10) (9) (9) (10) (8)
Serous
Other malignant
379 • 67 (11) 304 • 38 (14) 40.5 • 3.6 e (15) 38.3 • 7.6 (8) 7.4 • 1.6 (15) 3.35• 0.74 (15) 0.37• 0.21 (8)
269 • 70 (9) 277 • 98 d (11) 24.0 • 4.9 e (10) 22.7 • 10.2 (7) 9.1 • 1.9 (11) 3.05• 1.14 (12) 1.48• 0.52 (9)
0,255 0.002 0.037 0.616 0.232 0.217 0.273
" Assay procedures are described in Materials and methods. Values are expressed in nmol min 1mg cytosolic protein 1 except for catalase (gmol m i n i mg- 1), superoxide dismutase (units/nag) and non-protein thiols (nmol/ml cytosol) b Values given are the mean _+ the standard error, and the number of samples (n). The majority of malignant ovarian tissue was of the serous type The statistical significance of the difference in means across the four tissue types was determined using one-way unbalanced fixedeffects analysis of variance d Means for normal and benign tissue are significantly different (P < 0.05 by the Tukey multiple comparisons method) from other malignant tissue e Mean for serous tissue is significantly different (P
