Main Drug-Metabolizing Enzyme Systems in Human Breast Tumors and Peritumoral Tissues1

ICANÅ’R RKSKARCH 53. 3541-3546. August 1. 1993] Main Drug-metabolizing Enzyme Systems in Human Breast Tumors and Peritumoral Tissues1

Nicolas Albin, Liliane Massaad, Caroline Toussaint, Marie-Christine Mathieu, Jackie Morizet, Orlando Parise, Alain Gouyette, and Guy G. Chabot2

DÃ©partement Je Pharmaca-ttmcologie el PharmamgÃ©nÃ©tiiiue(INSERM U 140 and CNRS URA 147} Â¡N.A.. L. M., C. T.. J. M.. O. P.. A. G.. G. C. C.Â¡.and DÃ©partement d'Anatimui-Patholiixie Â¡M-C.M.l Institut Gustavt-Roussy, V4805 Villejiiif Cedex. France

ABSTRACT thus a very important health problem because of its considerable economic and social implications ( 1). In an attempt to better understand breast tumors sensitivity or resis The most active anticancer drugs used in breast cancer include tance to anticancer drugs, the main drug-metabolizing enzyme systems cyclophosphamide, methotrexate, 5-fluorouracil. doxorubicin, and were evaluated in both breast tumors and their corresponding peritu- vincristine (2). Cisplatin has also shown activity in the treatment of moral tissues in 12 patients. The following enzymes were assayed by Western blot: cytochromes P-4SO (1A1/A2, 2B1/B2, 2C8-10, 2E1, 3A4); advanced disease, especially in nonpretreated patients (3). Although glutathione .S'-transferases (GST-a, -u, and -TI); and epoxide hydrolase. the most frequently used drug combinations CMF or CMFP (cyclo The activity of the following enzymes or cofactor were determined by phosphamide, methotrexate, 5-fluorouracil, prednisone) can reduce spectrophotometric or fluorometric assays: GST; total glutathione; I 1)1'- mortality in women under 50 years old (4), women who relapse during glucuronosyltransferase; ÃŸ-glucuronidase; sulfotransferase; and sulfa or soon after adjuvant systemic therapy (within 6-12 months after its tase. Results showed the absence of all probed cytochromes P-450 in both cessation) have a poor prognosis. These patients do not respond to the tumoral and peritumoral tissues. GST activity was significantly (P < 0.05) same systemic therapy used as adjuvant (5). In contrast, patients who higher in tumors (mean Â±SD, 399 Â±362 nmol/min/mg) than in corre develop mÃ©tastasesafter a long cessation of adjuvant treatment appear sponding peritumoral tissues (86 Â±67). The GST isoenzymes GST-u and to respond to the same therapy, especially if the sites of recurrent GST-IT (determined by immunoblotting) were also higher in tumors than in corresponding peritumoral tissues (3- and 5-fold, respectively). Both disease are local, regional, or in soft tissue (6). GST-u and GST-m levels were significantly correlated with GST activity. To better understand the biological characteristics of breast tumors GST-a was not detected in either tumoral or peritumoral tissues. Glu and also the response or resistance to chemotherapy in this disease, we tathione levels in tumors (22 Â±23 nmol/mg protein) were not statistically have explored the main drug-metabolizing enzyme systems in breast different from peritumoral tissues (11 Â±12). Epoxide hydrolase was ex tumors and peritumoral tissues. These drug-metabolizing systems are pressed at similar levels in tumors and peritumoral tissues. The glucu- known to play an important role in the susceptibility of organs or ronide-forming enzyme UDP-glucuronosyltransferase was 5-fold lower in tissues to toxic effects of drugs or other xenobiotics and could also tumors (0.1 Â±0.2nmol/h/mg) than in peritumoral tissues (0.5 Â±I), whereas influence tumor response to anticancer agents in vivo (7, 8). Although the opposite was observed for the hydrolytic enzyme ÃŸ-glucuronidase, recent progress has been made in human breast cancer concerning which was 6-fold higher in tumors (736 Â±1392 nmol/h/mg) compared to some enzymes involved in drug metabolism (9, 10), no information is peritumoral tissues (125 Â±75). No difference was noted between tumoral presently available concerning many other important enzymatic sys and peritumoral tissues for sulfotransferase ( I Â±2 nmol/h/mg), but the corresponding hydrolytic enzyme (sulfatase) was 2-fold higher in tumoral tems in this tumor type. It was therefore of interest to assess breast tumors main phase I P-450s' and phase II enzyme systems involved tissues (14 Â±15 nmol/h/mg) than in peritumoral tissues (6 Â±2). In con clusion, several differences were observed between human breast tumors in drug metabolism (GST isoenzymes, LJDP-GT. and sulfotrans and peritumoral tissues for many conjugating enzymes (GST-u, GST-m, ferase), which are involved in functionalization and conjugation re and UDP-glucuronosyltransferase) and hydrolytic enzymes (sulfatase and actions, respectively. We also assessed GSH content and the hydro ÃŸ-glucuronidase). These noteworthy differences between tumoral and per lytic enzymes EH, ÃŸ-glucuronidase. and sulfatase. itumoral tissues with regard to their main drug-metabolizing enzymes In this report, we present the comparison of the main drug-metab could play a role in the relative drug sensitivity or insensitivity of human olizing enzyme systems in human breast tumors and their correspond breast cancer tissues to chemotherapeutic agents and could be potential ing peritumoral tissues. Our results showed significant qualitative and targets for chemotherapeutic interventions. quantitative differences between tumoral and peritumoral tissues, for many enzymatic pathways. These differences between breast tumors INTRODUCTION and peritumoral tissues with regard to drug-metabolizing enzyme systems could play a role in the relative drug sensitivity or insensi Breast cancer is the leading cause of death in women aged between tivity of breast cancer tissues to chemotherapeutic agents. 35 and 55 years and. after cardiovascular disease, it is the second most common cause of death in women older than 55 years. During the past 20 years, both earlier diagnosis and adjuvant systemic treatment have MATERIALS AND METHODS been able to increase disease free and overall survival of most pa Chemicals and Enzymes tients. The next progress in this disease could be expected from new 5.5'-dithiobis(2-nitrobenzoic acid), NADPH, 4-methylumbelliferone, 4-m- active agents or alternative modalities of treatment. Breast cancer is ethylumbelliferone sulfate. 4-methylumbelliferone glucuronide. reduced GSH. l-chloro-2.4-dinitrobenzene. ÃŸ-glucuronidase, and sulfatase were purchased from Sigma Chemical Co. (St. Louis, MO). PhÃ©nobarbitaland 3-methylcholan- Received 2/4/93; accepted 5/24/93. threne were purchased from Specia (RhÃ´ne-Poulenc, Paris, France) and from The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Aldrich (Milwaukee, WI), respectively. All other chemicals were of the highest 18 U.S.C. Section 1734 solely to indicate this fact. purity available from standard commercial sources. 1This work was supported by the Fondation pour la Recherche MÃ©dicale,the Institut National de la SantÃ©etde la Recherche MÃ©dicale(INSERM). and thÃ©CentreNational de la Recherche Scientifique (CNRS). 1The abbreviations used are: P-450. cytochrome P-450; CDNB. l-chloro-2,4-dinitro- 2 To whom requests for reprints should be addressed, at Institut Gustave-Roussy. benzene; GST, glutathione Â¿'-transl'erase;GSH, glutathione; EH. epoxide hydrolase; UDP- DÃ©partementde Pharmaco-toxicologie et PharmacogÃ©nÃ©tique.Pavillonde Recherche 2. GT. UDP-glucuronosyltransI'erase; GST-a. basic GST; GST-ir, acidic GST; GST-u., neu 94X05 Villejuif Cedex. France. tral GST. 3541

Human Tissues Antigens and Antibodies

Liver samples were provided from the Necker hospital tissue bank (Paris, In a previous study it was demonstrated that antibodies directed against rat France). Breast tissues were kindly provided by Dr. M-C. Mathieu (Gustave- or human antigens could recognize the corresponding mouse liver proteins Roussy Institute. Micropathology Laboratory, Villejuif, France). Breast tumors (15). and their corresponding peritumoral tissues were obtained from 12 different P-450. In this study we used the P-450 gene nomenclature based on the patients. All tissues were obtained during surgical excision of the tumor done updated list published by Nebert et al. (16). Polyclonal antirat P-450 1A1/1A2 and antirat 2B1/B2 were prepared in Dr. Beaune's laboratory (INSERM U 75, before chemotherapy. Peritumoral specimens were taken in a macroscopically Paris, France) and checked against Dr. F. P. Guengerich's preparations of P-450 noncancerous region of the resected tumor pieces (tumorectomy or mastecto my). Specimens were fro/en within 30 min and stored at -80Â°C to allow (Vanderbilt University, Nashville, TN). The polyclonal anti-rat P-450 2E1 was preservation of cellular enzymes. Part of each tissue sample was reserved for kindly provided by Dr. C. S. Yang (17). Monoclonal anlihuman P-450 3A4 was prepared as previously described (18). Polyclonal antihuman P-450 2C8-10 histopathological examination and the remainder was used for this investiga tion. was described by Shimada et al. (19). EH. Human epoxide hydrolase and their corresponding polyclonal antibod ies were previously described by Beaune et al. (20) and checked against Dr. Mouse Tissues Guengerich's preparations. GST. The polyclonal antihuman GST-a. -fi. and -n were purchased from In some experiments, we used livers from C57BI/6 mice pretreated with phÃ©nobarbitalor with 3-methylcholanthrene as positive references. A pool of Bioprep (Stillorgan, Dublin. Ireland). mouse liver was used as an internal standard in each enzymatic assay described Preparation of Samples for Enzymatic Assays below to ensure reliability of the data. Tissues were homogenized in 2 mm Tris-HCl (pH 8.1 ), 230 imi mannitol. Preparation of Samples for Western Blot Analysis and 70 rriMsucrose, using a Polytron homogenizer. These homogenates were used for the determination of total GSH and for the following enzymatic Mouse or human livers, breast tumor, and peritumoral adjacent breast tissue activities: UDP-glucuronosyltransferase; ÃŸ-glucuronidase; sulfotransferase; were quickly excised and rinsed in ice-cold 1.15% potassium chloride solution. and sulfatase. GST activity was assayed on cytosols. To minimize interday Tissues were minced with scissors and homogenized in phosphate buffer (0.1 assay variation, patient samples were run in batch for the same enzymatic M;pH 7.4), containing EDTA (0.1 m.M).After sonication of human and mouse assay. Pooled mouse liver was also used as an internal standard in each tissues, microsomes and cytosols were prepared by differential centrifugation enzymatic assay described below to ensure reliability of the data. as previously described (II). Microsomal pellets were suspended in phosphate buffer (0.1 mvi; pH 7.4) containing 20% glycerol and 10 m.MMgCl2. and then GST Activity stored at -80Â°C. GST was assayed as described by Habig et al. (21) using CDNB as sub strate. Formation of the CDNB-GSH conjugate by cytosols was measured Western Blot Analysis continuously in a spectrophotometer at 340 nm. The results were expressed in Microsomal or cytosolic proteins (50 fig) were separated by electrophoresis nmol of CDNB conjugated/min/mg of cytosolic protein. on sodium dodecyl sulfate polyacrylamide gels as described by Laemmli (12). Resolved proteins were electrotransferred to nitrocellulose sheets which were Total GSH Concentration probed with antibodies and stained as described previously (13, 14). The The sum of the reduced (GSH) and oxidized forms of glutathione was optical density of each stained band was determined by scanning with a determined in tissue homogenates using the method of Akerboom and Sies densitometer (Venon. PHI, Paris, France). The amount of each isoenzyme was (22). The results were expressed as nmol/mg of protein. expressed as arbitrary units/ng of microsomal or cytosolic protein. Arbitrary units were comparable for the different tissues studied in the same species but UDP-Glucuronosyltransferase Activity were not comparable between species. The threshold of protein detection for all the antibodies used was about 1% of the concentration of each isoenzyme The activity of this enzyme was assayed by fluorometry in tissue homoge measured in the liver. For all experiments we used positive controls (human nates using 4-methylumbelliferone as substrate according to El Mouelhi et al. liver, mouse liver, and liver from mouse pretreated with phÃ©nobarbital or (8). The results were expressed as nmol of glucuronide formed/h/mg of protein 3-methylcholanthrene). (nmol/h/mg).

Table 1 Patient and breast tumor characteristics PatientNo.123456789101112"

rupture'+

++-++

++ +-+

++ +-++

TNM classification widtlrfnrmn|^. X h Tumor volume wasAge(yrs)765872544359497638X28244deteninw-1by two-dimensionalVolume*(cm3)343.64.44.41.441316212.92514measurements(cm) using theHistology'IDCIDCIDCIDCIDCIDCIDCIDCIDCIDCIDCIDClengthcTumorHistologicalgrade17IIIIIIIIinnnunnininCapsularNodes' ' Histological grade; IDC. invasive.TNM"T2NoMoT2N0Mâ€žT,Nâ€žMâ€žT2NoMâ€žT,Nâ€žMâ€žT2Nâ€žMâ€žTiNoMoTiN(jM()T2Nâ€žMâ€žT|Nâ€žMâ€žT2N0M0TÂ¡NUM0nined ductal carcinoma. ''Modified Scartf and Bloom grading: 1. Clin. Oncol. 5: 1378-1386, 1987. ' +, histologicallylymphnodemetastasis:-, absenceoflymphnodemetastasis. ^Capsular rupture of lymph nodes: +, presence; -, absence. 3542

ÃŸ-Glucuronidase Activity Table 2 Glulathione S-transferase enzvmtÃticactivity; glutalhitwe S-transferase oenzvme.Ã¯,and GSH foncfntrittion in human breast tumors und perilumvral tixsues

The activity of this hydrolytic enzyme was assayed in tissue homogenates tissue86 using 4-methyl umbelliferone glucuronide as substrate according to El Mouelhi GST activity" Â±362*(12)''54 Â±67'' (12) el al. (ÃœÃ•.Resultswere expressed as nmol/h/mg of protein (nmul/h/mg). GST-crr ND GST-M1' Â±70*(12) I0Â±24*(12) Sulfotransferase Activity GST-Tr''GSH 61 Â±69*(I2) 22 Â±24*(12) 11 Â±12'' (12)PNS1 contentTumor399 22 Â±23'' (12)Peritumoral Sultbtrunsferase was assayed in tissue homogenates using a .V-phosphate- " GST activity was assayed spcclrophotometrically using I-chloro-2.4-dinitro-benzene adenosine-.V-phosphosulfate generating system, in the presence of 4-methyl- as substrate. '' Mean Â±SD. in nmol/min/mg of protein. umbelliferone to obtain 4-methylumbelliferone sulfate, and sodium sulfite to ' Numbers Â¡nparens. number of observations. inhibit hydrolysis of the formed sulfate conjugate as described by El Mouelhi '' Wilcoxon test between tumoral and corresponding peritumoral breast tissues. et al. (8). The results were expressed in nmol/h/mg of protei". '' Determined by Western blot. 1 ND. not detectable. Sulfatase Activity * The amount of each isoenzyme is expressed as arbitrary units/ng of cytosolic protein. h GSH content (mean Â±SD). determined spectrophotometrically expressed in nmols of Sulfatase was measured in the presence of 4-methylumbelliferone sulfate total GSH/mg protein. ' NS. not significant. using tissue homogenales. according to El Mouelhi et al. (8). Results were expressed as nmol/h/mg of protein.

Protein Assay tissue. Two patients expressed GST-/Min tumors but not in peritumoral tissues, and 1 patient had low levels of this isoenzyme in peritumoral Protein concentrations of microsomes. cytosols. and homogenates were tissues only. The comparison between tumoral and peritumoral tissues determined by the bicinchoninic acid assay (23) using a commercial prepara showed that GST-/J, was 5-fold higher in tumors than their correspond tion (Pierce BCA Protein Assay Reagent). ing peritumoral tissues (P < 0.01). Statistical Analysis GST-IT. This isoenzyme was expressed in all tumoral and peritu moral tissues. GST-Tr expression was 3-fold higher in tumors than in The level of significance was determined using the Wilcoxon test for paired peritumoral tissues (P < 0.01). A typical Western blot depicting the data between human tumors and their corresponding peritumoral tissues. The immunoquantitation of GST-ir in both tissues is presented in Fig. 1. correlation between GST activity determined by spectrophotometry and GST-Tr and -jj. determined by immunoblotting was determined using the Spearman's Correlation between GST Isoenzymes and GST Enzymatic Assay. A significant correlation was found between the GST activity coefficient of rank correlation. For these tests the level accepted as significant determined by spectrophotometric assay and both GST-/J. (r=0.69) was P < 0.05. and GST-Tr (r = 0.52) determined by immunoblotting (P < 0.01; Spearman's coefficient of rank correlation test) (Fig. 2). RESULTS Total GSH. Concentrations of total GSH in tumors and peritu Characteristics of Breast Tumor Samples moral tissues are presented in Table 2. Although GSH mean concen tration was 2-fold higher in tumors compared to peritumoral tissues, The 12 patients entered on this study had a median age of 58.5 years it did not reach statistical significance. Of interest was that the GSH and a tumor volume ranging from 1.4 to 34 cm3 (Table 1). All breast content in either tissues correlated significantly with GST-IT expres tumors were histologically confirmed as invasive ductal carcinomas. sion (P < 0.01). The TNM classification and the tumor histolÃ³gica! characteristics are presented in Table 1. Epoxide Hydrolase

Cytochromes P-450 EH was expressed at similar levels in both tissue types tested (Table 3). A typical Western blot depicting the immunoquantitation of EH in Western blot immunoquantitation of the main P-450 isoenzymes tumors and peritumoral tissues is presented in Fig. 3. was performed in breast tumors and peritumoral adjacent breast tis sues. As positive controls, human and mouse livers were also ana Glucuronide System lyzed. None of the probed P-450s (including 1A1/1A2, 2B1/B2, 2C, 2E1, UDP-Glucuronosyltransferase. UDP-GT activity was detected in and 3A) was expressed in either tumoral or peritumoral tissues (n = 4 patients in both tumors and peritumoral tissues and in 1 patient in 12). In parallel experiments, all of these P-450s were detected in peritumoral tissue only. Seven patients did not show detectable levels mouse and human livers, as expected. of this enzyme in either tumors or peritumoral tissues. The activity of this conjugating enzyme was significantly lower in tumors (5-fold) Glutathione System compared to peritumoral tissues (P < 0.01) (Table 3). ÃŸ-Glucuronidase. ÃŸ-Glucuronidaseactivity was detected in all GST Activity. The GST activity in tumoral and peritumoral tissues tissues studied. This enzymatic activity was markedly higher (6-fold) is presented in Table 2. There was a significant difference in CDNB- in tumors compared to peritumoral tissues (P < 0.01) (Table 3). GST activity (representing total GST activity) between tumoral and peritumoral tissues, with a 5-fold higher GST activity in tumors (P < Sulfate System 0.01). Glutathione S-Transferase Isoenzymes. Western blot immuno Sulfotransferase. This conjugating enzyme was detected in 6 of quantitation of GST isoenzymes is presented in Table 2. the 12 tumors and in 5 of the 12 peritumoral tissues investigated GST-ot. This isoenzyme was not expressed in any of the tumoral (Table 3), but no significant difference was observed between the and peritumoral tissues studied (n = 12). tissue types. GST-fi. This isoenzyme was expressed in 8 of the 12 tumoral Sulfatase. This hydrolytic enzyme was detected in all tissues stud tissues studied and in 7 of the 12 peritumoral tissues. In 3 patients, ied. Sulfatase activity was 2-fold higher in tumors (P < 0.05) com GST-/J. was not expressed in either the tumoral or the peritumoral pared to corresponding peritumoral tissues (Table 3). 3543

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Fig. I. Western blot of human and mouse cytosols developed with polyclonal anti-human GST-7T. Lane /. human liver (50 Â¿igprotein); iMne 2, mouse liver (25 Â¿Â¿gprotein);Lane 3, tumor 1 (50 fig protein); Lane 4, perilumoral tissue 1 (50 /ig protein); Lane 5, tumor 2 (50 jig protein); Lane 6, peritumoral tissue 2 (50 fig protein); Ume 7. tumor 3 (50 jzg protein); Lane 8* peritumoral tissue 3 (50 /Ag protein); Lane 9, tumor 4 (50 jxg protein); Lane 10. peritumoral tissue 4 (50 p.g protein); Lane Â¡I, mouse liver (25 Â¿ig protein); Lane Â¡2.human liver (50 jxg protein).

concluded that P-450C21 could be closely related to the ductal dif

240 ferentiation in breast neoplasie transformation. The absence of the main P-450s in breast tissues indicates this tissue would be deficient ^200 in performing P-450-dependent oxidative reactions such as hydroxy- _o a E 160- lation, epoxidation, or dealkylation, that may toxify or detoxify certain 0> 1 drugs. If drugs are not metabolized by other enzymes, e.g., conjugat | 120 ing enzymes, the retention of drugs within breast tumor cells would be likely, thus favoring possible cytotoxic effects. 80- The glutathione system, another major enzymatic pathway involved 40- in the detoxication of exogenous compounds (28) was also studied in tumoral and peritumoral breast tissues. The concentration of the var 0.2 04 0.6 0.8 ' 1.2 ious GST isoenzymes in normal and tumoral tissues is important GST activity (nmol/min/mg x IO"3 ) because these enzymes play a central role in the detoxication of many toxic electrophilic compounds, including carcinogens and cytotoxic Fig. 2. Correlation between GST activity (determined by spectrophotometry analysis) and GST-TT(determined by Western blot analysis) in human breas! lumors and peritumoral drugs. Given the great diversity of their functions, several authors (29, tissues (r = 0.52; P < 0.01; Spearman's coefficient of rank correlation test). 30) have postulated that the variability in the expression of these enzymes could be a factor in the susceptibility of various tissues to DISCUSSION toxins and carcinogens and suggested that they may be used as po tential prognostic markers in carcinogenesis. Increased GST activity In an attempt to better understand human breast tumors sensitivity and increased tumoral GSH content were associated with resistance to or resistance to anticancer drugs, we assessed the main drug-metab cytotoxic agents (31). olizing enzyme systems in both tumoral and adjacent peritumoral The cytosolic GST enzymes in human have been divided into three tissues. The drug-metabolizing enzyme systems studied are known to distinct groups which are commonly referred to as basic (a), neutral influence markedly the sensitivity of organs or tissues to the cytotox- (/j.), or acidic (TT)transferases, according to their isoelectric points icity and/or carcinogenicity of drugs or other xenobiotics, and they (32). In the present study, we did not detect GST-a in either tumoral could also determine tumor response to anticancer agents in either or peritumoral breast tissues. This finding is in accordance with a detoxication or toxic effects of these drugs (7, 8). Adjacent breast previous study (10) that also showed the absence of GST-a in 43 peritumoral tissues were chosen as references for human breast tu- primary human breast cancer samples tested. These data suggest that moral tissues because the activity and the expression of drug-metab GST-a may not play a predominant role in the detoxication of organic olizing enzyme systems may vary greatly between individuals, as a hydroperoxides in either tumoral and peritumoral tissues. function of genetic, environmental, and physiological factors (24). GST-/* has been shown to be genetically polymorphic and is not Cytochromes P-450, a multigene superfamily of microsomal hemo- expressed in 40-50% of the human population (33). In our small proteins, play a key role in the biotransformation of a wide variety of xenobiotics and are involved in their detoxication or toxic effects. P-450 isoenzymes are often involved in the biotransformation of Table 3 Conjugating and hydrvlytic enzymes in human breast lumors and peritumoral anticancer agents, and these enzymes are sometimes required for the tissues expression of anticancer activity, e.g., in the activation of cyclophos- EnzymesEpoxide tissue49Â±30(11) phamide (7, 25). In the present study, we did not detect any of the hydrolase" (11)' UDP-GT' 0.1 Â±0.2 (12) 0.5 Â±1 (12) probed P-450s (1A1/1A2, 2B1/B2, 2C, 2E1, and 3A4) in tumoral or ÃŸ-Glucuronidase1* 736 Â±1392 (12) 125 Â±75(12) peritumoral breast tissues. Pontin et al. (26) have determined the Sulfotransferase' lÂ±2 (12) 1 Â±2 (12) cytochrome P-450dbl phenotypes in malignant and benign breast Sulfatase'Tumor50Â±48* 14Â±15 (12)Peritumoral 6Â±2 (12)PNSd disease and have shown that breast cancer cases differed significantly " Determined by Western blot. Expressed as arbitrary unit/ng of protein. * Mean Â±SD. from the benign controls in their debrisoquine phenotype, as 10% of ' Numbers in parens. number of observations. The number is 11 for EH because of patients were poor metabolisers compared with none of the controls. insufficient tissue for one patient. Sasano and Sasano (27) have studied the cytochrome P-450C21 and d NS, not significant. * Enzymatic activities were assayed in tissue homogenates as described in "Materials they demonstrated that in histologically normal breast, this P-450 was and Methods." They were expressed in nmol/h/mg protein. exclusively detected in secretory tubules and ducts; these authors ^Wilcoxon test between tumoral and peritumoral breast tissues. 3544

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Fig. 3. Western blot of human and mouse microsomes developed with monoclonal anti-human EH. Lane I. phenoharbital-indueed mouse liver (20 jig protein); Lane 2. human liver (20 (ig protein); Lane 3, tumor I (50 (ig protein);Lane 4. peritumoral tissue I (50 (ig protein); Lane 5. tumor 2 (50 jig protein); Lane 6. pentumoral tissue 2 (50 fig pro tein); Lane 7. tumor 3 (50 (ig protein); Lane 8. peritumoral tissue 3 (50 (ig protein); Lane 9. tumor 4 (50 jig protein); Lane 10. peritumoral tissue 4 (50 jig protein); Lane II. human liver (20 (ig protein); Lane 12. phenobarbital-inducedmouse liver (20 (ig protein).

effective treatment modalities using a nontoxic "modulator" in com human population, this enzyme was found in 8 of 12 tumor samples studied (67%), in accordance with the polymorphism of this isoen- bination with a standard dose of antitumor agents so as to enhance the zyme. Our results are in agreement with those of Di Ilio et al. (33) effect of the cytostatic drugs without producing increased damage to who showed the presence of GST-/A in 18 of 38 primary human breast normal tissues (40). cancer samples (50%). These authors also demonstrated that the ab Epoxide hydrolase is thought to play a protective role with respect sence of GST-fj. results from a deletion of the GST-jn gene, which to metabolically produced epoxides from xenobiotics involved in provides an explanation for the total loss of the protein in the breast carcinogenesis and cytotoxicity (41). In our study, no difference was tissues of some patients studied. observed for EH between tumoral and peritumoral tissues. Since tu In agreement with other reports (34, 35), breast tumoral tissue had mors expressed EH at the same levels than peritumoral tissues, it is higher levels of GST-ir than their corresponding peritumoral tissues. expected that the two tissue types would be similarly vulnerable to GST activity in tumoral tissues was also well correlated with GST-IT epoxides. content. Recent studies have shown that the activity of the anionic No information is presently available concerning the glucuronide GST isoenzyme is increased in both rat preneoplastic hepatocyte and sulfate pathways in breast tissues. Our data showed significant nodules and Adrr MCF-7 cells (36, 37) and that the human form of the differences between tumoral and peritumoral tissues for these path anionic GST, GST-IT, is increased in multidrug-resistant Adrr MCF-7 ways. For the glucuronide system, UDP-GT was decreased, whereas cells when compared with the parental cell line (WT MCF-7) (38, 39). ÃŸ-glucuronidase was increased in tumoral versus peritumoral breast Hamilton et al. (40) showed an augmentation of Adriamycin, mel- tissues: this interplay of conjugating and hydrolytic action could favor phalan, and cisplatin cytotoxicity in drug-resistant ovarian cancer cell drug retention in tumoral tissues, since the low synthesis of the glu lines by buthionine sulfoximine-mediated glutathione depletion. curonide conjugate coupled to the high ÃŸ-glucuronidase activity could Buthionine sulfoximine is a specific inhibitor of the enzyme lead to drug retention. The glucuronidation pathway was observed for T-glutamyl cysteine synthetase, which is a key enzyme in the synthesis doxorubicin analogues and mitoxantrone (37, 42, 43). Also notewor of GSH (40). These results provide possibility of modulating or cir thy was the high sulfatase activity in tumors compared to peritumoral cumventing resistance to clinically useful drugs, thus providing more tissues: this hydrolytic activity could also favor drug retention in tumor by splitting sulfate conjugates. Consequently, these drug-me tabolizing pathways could favor a greater drug retention and perhaps Table 4 Summary of the comparison of human breast tumors and peritumoral tissues with regard to main drug-metabolizing enzyme systems an increased sensitivity of breast tumoral tissues to drugs metabolized via the glucuronide and/or sulfate pathways. EnzymesCyt tissueND* Peritumoral P-450"Epoxide ND*+ Taken together, the results presented in this article provide note worthy new information on the major drug-metabolizing enzyme sys hydrolaseCDNB-GSTGST-aGST-(iGST-TTGSHUDP-GTSulfotransfera.seÃŸ-GlucuronidaseSulfataseTumor++ + + + ++ND* tems in breast tissues, and they have indicated some similarities but ND*+++++ also many differences between tumoral and peritumoral tissues (sum ++ + +++ marized in Table 4). The similarities are the absence of P-450s in +-1- breast tumoral and peritumoral tissues. Intertissue differences were ++ + + + + +++++++ essentially due to the conjugating and hydrolytic enzyme levels or ++ activities which were markedly different between tumoral and peri + +PNSC^.Ol''<0.01rf<0.01<(NSC<0.05dNSC<0.01d<0.05dtumoral tissues. It is of interest to mention that similar changes in "Cyt P-450. Cytochromes P-450: IA1/A2; 2B1/B2; 2C8-10; 2E1; 3A4. drug-metabolizing enzyme systems are observed in multidrug resis * ND. not detectable. ' NS. not significant. tant tumor cells, i.e., decreased P-450s and increased activities of '' Wilcoxon test between tumoral and peritumoral breast tissues. several drug-conjugating enzymes (37, 44). 3545

In conclusion, breast tumoral and peritumoral tissues were signif 20. Beaune, P. H.. Cresteil, T. Flinois. J. P.. and Leroux, J. P. Hydrophobie chromalog- icantly different for many drug-metabolizing enzyme systems in raphy: a one step method for purification of human liver microsomal epoxide hydrolase. J. Chromatogr. Biomed. Appi.. 42ft: 169-176. 1988. volved in either toxication or detoxication of drugs. These intertissue 21. Habig, W. H.. Pahst, M. J., and Jakoby, W. B. Glutathione .S'-transferases. The first differences could intluence the susceptibility of breast tissues to an- enzymatic step in mercapturic acid formation. J. Biol. Chem., 249: 7130-7139, 1974. 22. Akerrxxim, T. P., and Sies, H. Assay of glutathione. glutathione disulfide. and glu- ticancer drugs and could partly explain breast tumors sensitivity or tathione mixed disulfides in biological samples. Methods Enzymol., 77: 373-384, resistance to chemotherapy. 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Nicolas Albin, Liliane Massaad, Caroline Toussaint, et al.

Cancer Res 1993;53:3541-3546.

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