Carcinogen Activation by Human Uterine Enzymes*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 9, No. 3 Copyright 1979, Institute for Clinical Science, Inc. Carcinogen Activation by Human Uterine Enzymes* JOH N C. M. TSIBRIS, Ph.D. Department o f Obstetrics and Gynecology, University o f Florida College o f Medicine, Gainesville, FL ABSTRACT Three topics are briefly review ed relating to carcinogenesis of estrogen responsive tissues: (a) enzymology of benzo(a)pyrene activation by hum an tissues, (b) microsomal activation of estrogens to estrogen arene oxides and (c) estrogen and progesterone receptor studies in endometrial carcinoma. The following working hypothesis is stated on the etiology of gynecologic tumors: Environm ental chemicals, such as cigarette smoke, polycyclic and polyhalogenated hydrocarbons, etc., induce special forms of cytochrome P-450 monooxygenase and related enzyme systems which can activate endogenous or prescribed estrogens and non-steroid antiestrogens to act as initiators and/or promoters of neoplasia in estrogen-dependent organs. The role of estrogen receptors is perceived as a homing device or cellular Trojan H orse for these activated estrogens. Introduction In recent years it has become apparent that the majority of hum an cancers are probably caused by environm ental chem icals, natural or synthetic, rather than by viruses or radiation and are therefore ultim ately preventable.5 T here is also a great deal of evidence to show that many chemical carcinogens, such as polycyclic aromatic hydrocarbons, require metabolic activation, m ainly by m icrosom al e n zymes, to highly reactive electrophiles w hich bind covalently to desoxyribonucleic acid (DNA), proteins, etc. and thus exert their carcinogenic and mutagenic effects.19 * Supported by grant No. CA from the National Cancer Institute, DHEW and Institutional grant NO from the American Cancer Society. Carcinogenesis appears to be a multistep process and it is probable that various environm ental chemicals act at different steps in this process. One of the best studied models in this respect is the socalled two-stage carcinogenesis system of m ouse skin in w hich two d istin ct stages have been identified, namely, initiation and promotion.6,29,3 Cancer can be produced by a combination of a single exposure to a carcinogen (initiator, such as benzo(a)pyrene), in a dose so low that it would normally not cause cancer, with a subsequent prolonged exposure to very small am ounts o f an agent (promoter, such as a phorbol ester, phénobarbital or saccharin) which alone is not carcinogenic. A ccording to the m odel, a single exposure to a high dose of the in itiator can cause cancer and the initiation /79/ $01.20 Institute for Clinical Science, Inc.

2 CARCINOGEN ACTIVATION 237 step leading to tum or form ation is irreversible. In contrast, the action of promoting agents (at an early stage) is reversible; they do not seem to bind covalently to cellular macromolecules and are not m utagenic.6,29,30 Benzo(a)pyrene (BP) is a very potent carcin o g en ic polycyclic hydrocarbon commonly found in the environm ent as a result of pyrolysis of organic compounds. BP is first converted to epoxides, arene oxides, by monooxygenases (cytochrome P-450 enzym e system s). T h ese oxygenated interm ediates can bind covalently to DNA, ribonucleic acid (RNA) and proteins24 and are sterospecifically hydrated to dihydrodiols by epoxide hydrase,2 conjugated with glutathione by glutathione epoxide tran sferase11 or nonenzym atically converted to at least four phenols and three quiñones. BP-phenols, and to a lesser ex ten t the o th er oxygenated form s of BP, can be c o n ju g ated as glucuronides14*27 or sulfates. The most exciting recent discovery in BP m etabolism and carcinogenesis was the realization that a secondary m etabolite of BP, a diol-epoxide, may be the predom inant carcinogenic form of BP31 (and references therein). T he m ost com m on source of BPm etabolizing enzymes is rodent liver and lungs, b u t th e se enzym es (aryl h y drocarbon hydroxylase, etc.) have also b e e n found in hum an liv er, lungs, peripheral blood cells,8 placenta3 and fetal endocrine glands.23 The finding of aryl hydrocarbon hydroxylase in placenta is ot interest in connection w ith the occurrence of adenocarcinomas in females whose m others had taken diethylstilbestrol (DES) during pregnancy, the m etabolic activation of D E S 17 and its covalent b in d in g to DNA.4 F e ta l enzym es metabolizing BP23 could be potentially im portant in transplacental chem ical carcinogenesis and toxicity. Estrogens, the only aromatic steroids in the human body, have been suspected for a long time of playing a role in the etiology of gynecologic cancers. Similarities in the structures of estrogens, diethylstilbestrol and BP (see Discussion) prom p ted us27,28 to test w hether or not natural and synthetic estrogens could be activated in vitro by microsomal enzymes from rat liver to form estrogen arene oxides; nucleic acids were added as in vitro traps of the presum ably very unstable interm ediates. These experiments show ed that estrogens and especially estro n e can be a c tiv ate d by a form of monooxygenase (induced by 3- methylcholanthrene) which is specific for the oxygenation of aromatic molecules, thus supporting the hypothesis that an arene oxide of estrogens was formed. Earlier, Booth et al3 had alluded to the possible formation of estrogen epoxides, and re cently Num azawa and Nam bara20 proposed that estrogen arene oxides may be involved in the conjugation of estrogens w ith glutathione. Recently, L equesne and associates reported the chem ical synthesis of estrone arene oxides.25 F u rth er characterization of the estrogen complexes with DNA and proteins is in progress (see Discussion) but it was felt that it was important to test if such BP enzymes were present in adult hum an ovaries, uterus and placenta; the first results are described in this report. Materials and Methods (G-3H) Benzo(a)pyrene was purchased* and was purified before use by high pressure liquid chromatography (HPLC) as follows: DuPont 830 chromatograph and Zorbax-ODS (4.6 mm x 25 cm) column, linear 3 percent per min gradient from 20 to 85 percent methanol in water at 50, flow rate 2 ml p er min; fractions w ere collected directly off the column every m inute. The p la c e n ta was from a cig arette smoker who was delivered by Caesarian * From Amersham-Searle.

3 238 TSIBRIS section and was given m eperidine and prom ethazine during the four hours prior to delivery and atropin, sodium pentathal, nitrous oxide and succinyl chloride during surgery. The placenta was perfused with cold 1 percent KC1 and microsomes were isolated from the cotyledon tissue.1 The ovary was from an anovulatory patient and was inflamed. The myometrial sample was from the fundus of a hysterectomy specimen and the patient was in early luteal phase of her cycle. All tissues were homogenized! in 50 mm potassium phosphate buffer, ph 7.4, containing 20 percent glycerol, and the homogenates were first centrifuged at 800 x g for 10 min and then at 105,000 x g for 60 min to obtain the post-nuclear particulate ( microsomal ) fraction. Protein was determ ined by the Lowry m ethod using bovine serum albumin as standard. Incubation mixtures contained in a final volume of one ml: buffer, salts, etc. as previously described,28 microsomes (1.15 mg from the placenta or 0.94 mg from the ovary or 1.08 mg from the myometrium), 50 nanomoles 3H-BP (specific activity: 150 cpm per picomole BP for the placenta and 130 cpm for the other tissues) and as in d icated nicotinam ide adenine dinucleotide phosphate (NADPH) and NADPHregenerating system and 3 mm UDPglucuronic a c id. After shaking in subdued light for 30 min at 37, the incubation mixtures were extracted27 twice with one ml of acetone and two ml of ethyl acetate; the organic phases were taken to dryness at 40 under a stream of nitrogen, were dissolved in 50 fjl1 methanol and injected into the HPLC. Fractions were collected every 30 or 60 sec and counted in eight ml of Bray s solution. E ach graph was normalized to 2 X 106 total cpm. Results In figure 1 is shown a comparison of the m etabolism of BP by nearly equal amounts t With a Tekmar Tissumizer. } Ammonium salt, Sigma Chemical Company. of microsomal proteins from a human placenta, an inflamed ovary (oophoritis) and a non-canerous myometrium. Under these reverse-phase chromatographic conditions, originally employed by Gelboin, Selkirk and associates and Jerina and associates,27 B P-dihydrodiols are eluted first (10 to 25 min), followed by BPquinones, BP-phenols (around 35 min) and finally by unm etabolized BP (ground 43 min). Incubations in the absence of added NADPH (A,B) are taken as control (blank) values; owing to the lim ited sample, there was no control run for the myometrium experim ents. Even though identification of each peak will require synthetic BPm etabolite standards and mass spectroscopic and/or NMR analyses, it is clear that all three samples m etabolized BP- UDPglucuronosyl transferase is found in microsom es and, in the presen ce of UDPglucuronate (UDPGA), it would convert B P -m etabolites (m ainly p h en o ls27) to w ater-soluble glucuronides. Thus, elimination of the BP-phenol and BP-quinone HPLC peaks after addition of UDPGA, strengthens the assignment of these peaks and indicates the presence of UD P-glucuronosyl transferase in the particulate (microsom al) fractions tested. W ith the possible exception of these placental microsomes (A' A"), both the ovarian (B ' B") and myometrial (C" C') sam ples contained UDPGA transferase activity; this is also supported by increased radioactivity in the corresponding waterlayers from experiments B" and C" after o rganic so lv en t extraction ( data not shown). Discussion High pressure liquid chromatography is an ideal m ethod to study BP metabolism because it can distinguish m etabolites form ed by m onooxygenases* epoxide hydrase(s) and by the three conjugating enzym es, nam ely, the transferases of UDPGA, glutathione and sulfates. Pelkonen21 used the fluorescent assay and re-

4 CARCINOGEN ACTIVATION 239 F ig u r e 1. High pressure liqu id chromatograms of acetone-ethyl acetate extracts of 3H-BP and its metabolites produced during incubations under air of human microsomes from placenta (A, A', A"), ovary (B, B', B") and myometrium (C', C"). NADPH is the source of electrons and UDP-glucuronate (UDPGA) is required for the glucuronidation of some BPm etabolites form ed in s itu ; unreacted BP, indicated by the dotted line, eluted at min. D etails on incubations and HPLC are given under M aterials and Methods. ELUTION TIME, MIN ported 12.2 ± 9.1 and 0.05 femtomoles of the fluorescent 3- and 9-phenols of BP formed per min per mg of protein from two groups of smokers and non-sm okers, respectively; Berry et al3 reported such activity from two placentas as 5.5 and 840 picomoles. From figure 1, one can calculate the BPm etabolizing activities of the three tissues tested by subtracting the NADPH from the +NADPH data; the results are 3.9, 6.1 and 12.6 picom oles of BPm etabolites (eluting before BP in the HPLC graphs) produced per min per mg of?ch2ch2nd Figure 2. Structures of some synthetic and natural polycyclic aromatic hydrocarbons. A, B and C, non-steroid antiestrogens, D, 17/3-estradiol, E, benzo- (a)pyrene, F, ethinyl estradiol, G, equilin and H, equilenin. R, represents a hydrogen atom or alkyl group. Q=CH ~ OH G HO o a OH

5 240 TSIBRIS protein by placental, ovarian and myometrial samples, respectively. Differences in specific activities may be due, in part, to genetic differences among various individuals and possibly reflect different d e grees o f sa tu ra tio n of the drug m etabolizing enzymes by drugs, cigarette smoke components, etc. Perhaps a better estimation of the m etabolizing capacity of microsomes could be obtained if the latter were freed by Sephadex chromatography from excess endogenous substrates (steroids, drugs, etc.) before incubation with BP. BP m etabolites covalently bound to microsomal proteins should also be included since they represent direct binding of BP arene oxides to nucleophilic amino acid residues. O n the other hand, prolonged exposure to some drugs, cigarette sm oke, etc. could also induce BPm etabolizing enzym es in the tissues tested. The finding th at hum an endocrine glands and estrogen target organs have the enzym es required to activate a potent aromatic carcinogen and, by extrapolation from our work with rat liver,28 estrogens elicit some new thoughts on the etiology of cancer of estrogen-responsive tissues. As seen in figure 2, there are many structural sim ilarities betw een estrogens (natural and prescribed), non-steroid antiestrogens and benzo(a)pyrene and some ofits phenol metabolites. Whereas there are also many similarities in the metabolism of BP (chosen as a representative compound among carcinogenic aromatic hydrocarbons) and of estrogens, there are also two important differences: (1) BP is an inducer of the activation enzym e system s (e.g. m onooxygenase, epoxide h ydrase) whereas estradiol and estrone are not, and (2) there is a w ell-characterized protein, the estrogen receptor, which specifically carries estrogens from the cytoplasm to the nucleus w hereas the existence of such specific carriers for BP and other polycyclic carcinogens is not yet certain.10 It is possible that a unique situation occurs w h en an e stro g e n -d e p e n d e n t organ is exposed to environm ental carc in o g en s as stated in the follow ing w orking hypothesis: E nvironm ental chem icals, such as c ig a rette sm oke, p olycyclic and polyhalogenated hydrocarbons, etc., induce special forms of cytochrome P-450 m onooxygenases and related enzym e systems which can activate endogenous or prescribed estrogens and non-steroid antiestrogens to act as initiators and/or prom oters of n eo p la sia in estro g en - dependent organs. The role of estrogen receptors is perceived as that of a homing device or cellular Trojan Horse which transports the activated estrogen to a specific site(s) on chromatin. Because of the instability and enzym atic detoxification of these estrogen interm ediates and DNA repair enzymes, the manifestation of hyperplasia or neoplasia could, in a sense, be an overdose effect. This hypothesis does not require that the activation of estrogens takes place in the uterus, ovaries, etc., although the finding of in situ activation of BP strengthens this thesis. It is possible that estrogens are activated in the liver, lung, kidney, etc., leak out of the organ and are specifically re- -COCHNH- I CH HO H esteróse HO H- HO Glu + or Gla F ig u r e 3. Proposed attachment of 1.2-arene oxide of estrone with the glutam ic or -y-carboxyglutamate sid e chain residue of a microsomal protein. Thermolysin and esterase digestion of the protein would liberate the amino acid and the triol derivative of estrone.

6 CARCINOGEN ACTIVATION 241 ta in e d by and cause h y p e rp la sia or neoplasia in estrogen target organs. W hereas DNA is generally accepted as playing a central role in chem ical carcinogenesis, it is possible that activated estrogens or BP could bind and, for example, slightly alter the fidelity of key proteins such as DNA polymerase, DNA repair enzymes, etc., resulting indirectly in the carcinogenic alteration of DNA after the first cell division. It seems likely that the binding to microsomal proteins of activated estrone or BP involves an ester bond as shown in figure 3. R ecent u n p u b lish ed experiments to characterize the 3H-hydrocarbonam ino acid adduct(s), after enzym atic hydrolysis of the proteins and isolation of the adduct(s) by HPLC, revealed that the hydrocarbon moiety of the adduct(s) was liberated after treatm ent w ith proteinase K or a-chym otrypsin or alkaline phosphatase (all three enzymes are serine esterases) b u t n o t w ith therm o ly sin, snake venom phosphodiesterase at ph 9 or buffer ph 9; therm olysin and pepsin are two proteases free of esterase activity.18 Such ester linkage would limit the amino acid moiety of the adduct to an aspartic, glutamic or -y-carboxyglutamate residue. The question w hether or not estrogens are carcin o g en ic (estro g en-can cer hypothesis, eloquently discussed12 by Hertz and Jensen) is still a matter of discussion.9,28 My hypothesis implies that estrogens per se are not initiators of carcinogenesis, although the role of some rep lacem en t estro g en s n eed s further evaluation and provides a biochem ical mechanism for the possible involvement of environmental inducers of activating enzym es in th e d e v e lo p m en t of gynecologic tumors. In my opinion, the human uterus is the ideal organ to test the existence of a possible link betw een the influence of steroid and peptide hormones on carcinogen activating enzymes and carcinogenesis, ow ing to its relatively easy accessibility and normal hormonal fluctuations during the menstrual cycle. Estrogen and progesterone receptors, which have been used successfully in deciding the course of therapy for breast cancer patients,16 will undoubtedly be useful in the previous studies, although in endom etrial carcinom a there is no strong correlation betw een receptors and the histological grade of the tumor.13,15,22 O ne of the technical difficulties encountered in such correlation studies is that, unlike the non-lactating normal breast which has no estrogen receptors, cycling and postm enopausal u teri have an uneven distribution of estrogen and progesterone receptors along the long axis of the endom etrium.26 Factors w hich could affect the concentrations of receptors found in endom etrial biopsies are the location of the endom etrial tumor, the day of the m enstrual cycle on which the biopsy is done, the am ount of norm al tissue removed and the dependence of the tumor steroid receptors on the day of the cycle. Use of the uteroscope and biopsy of tumor and adjacent normal tissue could alleviate some o f the difficulties. Acknowledgm ent Thanks are extended to my colleague, Kenneth R. Kellner, M.D., Ph.D., for his assistance with the placenta perfusion, William N. Spellacy, M.D. and other members of this Department for their help in clinical aspects of this project and many stimulating discussions. References 1. Be l l in o, F. L. and O saw a, Y.: Solubilization of estrogen synthetase from human term placental m icrosom es using detergents. J. Steroid Biochem. 9: , Be n t l e y, P., Sc hm assm ann, H., Sim s, P., and O e s c h, F.: Epoxides derived from various p olycyclic hydrocarbons as substrates of homogeneous and microsome-bound epoxide hydrase. A general assay and kinetic properties. Eur. J. Biochem. 69:97-103, B e rry, D. L., Z a c h a r ia h, P. K., S la g e, T. J., and JUCHAU, M. R.: Analysis of the biotransformation of benzo(a)pyrene in human fetal and placental tissues with high-pressure liquid

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