Chem. Res. Toxicol. 1994, 7, 696-701
696
Reaction of Lysine with Estrone 3,4-o-Quinone Katmerka Tabakovic and Yusuf J. Abul-Hajj* Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 Received May 6, 1994@ Reaction of lysine with estrone 3,4-o-quinone gave a complex mixture of products. Six compounds were isolated and identified using spectroscopic techniques. Among the reaction products isolated were 4-hydroxyestrone (2), 3-aminoisoestrone (3),3-(N-pentyl-5-amino)isoestrone (4), 1-lysylestrone 3,4-o-iminoquinone (5), and two dimeric products of 3,4-catechol estrone (6 and 7).
Introduction Elucidation of the mechanism(s1 underlying hormonal carcinogenesis caused by estrogens has been a major research objective. It has long been recognized that estrogens could induce tumors in rodents ( I ) . However, only during the last two decades has the potential of estrogens to induce tumors in humans been widely recognized (2-4). Although the carcinogenic mechanism(s) of estrogens is (are) not clearly understood, one mechanism currently under investigation is their metabolism to catechol forms with subsequent oxidation to the quinoneslsemiquinones. Studies in our laboratories have shown that 3,4-estrone quinone (3,4-EQI1was capable of inducing specific DNA damage in a human breast cancer cell line (5). These studies showed that 3,4-EQ is unique among o-quinones in that it causes exclusively single-strand DNA breaks1 alkali-labile sites. Furthermore, studies by Nutter et al. (6) provided conclusive evidence documenting the production of hydrogen peroxide, the hydroxyl radical, and the semiquinone of 3,4-EQ. Although production of reactive oxygen species (ROS) through redox cycling has been implicated in damage to macromolecules, including DNA, RNA, and proteins (71, the assignment of the actual ROS by 3,4-EQ-induced DNA damage awaits further study. Another potential mechanism which may be involved in the carcinogenicityltoxicity of estrogens involves adduct formation with DNA and/or proteins. Estrogen o-quinoneslsemiquinones and estrogen 1,2-epoxideshave been proposed to be responsible for estradiol's genotoxic activity. Tsibris and McGuire (8)proposed that binding of estrogens to nucleic acids involves an arene oxide intermediate since there was increased binding of estrogen to nucleic acids. However, these results have not been confirmed and are in conflict with the results obtained by Bolt and Kappus (9) and Numazawa et al. (10). Moreover, any attempts a t formation of nucleoside or DNA adducts with estrogen quinones or the ketotautomers of estradiol epoxides have not yet been successful.2 Thus, it is quite clear that while metabolic activation plays a role in estrogen carcinogenesis (11,121, covalent DNA binding with either the estrogen quinones or epoxides apparently does not take place.2 Even though * Author to whom correspondence should be addressed.
Abstract published in Advance ACS Abstracts, August 15, 1994. Abbreviations: 3,4-estrone quinone, 3,4-EQ; reactive oxygen species, ROS; glutathione, GSH. Y. J. Abul-Hajj, unpublished results. @
LeQuesne et al. (13) showed that 4a,5a-epoxy-l7Phydroxy-1-estren-3-one (ketotautomer of estradiol 4a,5aepoxide) induced transformation of BALBlc 3T3 fibroblasts, studies in our laboratory showed that the major pathway for irreversible binding of estrogens to proteins involves the estrogen o-quinoneslsemiquinones and not the estrogen arene oxides (14). Thus, it is quite possible that the carcinogenicityltoxicity of estrogens may be, in part, due to estrogen o-quinoneslsemiquinones which are capable of reacting with proteins. It has been well documented by us and others that glutathione and cysteine react with estrogen o-quinones by Michael addition, leading to the formation of thioether adducts (15,16). Furthermore, addition reactions between lysine and quinones have been implicated in several proteinquinone interactions. For example, the decrease in lysine content of casein following the incubation of caffeoquinone and chlorogenoquinone has been attributed to the addition of the +amino group of lysine to respective quinone moieties (17, 18). Recent studies in our laboratories showed that propylamine can react by 1,2- and 1,6-Michael addition to 2,3and 3,4-estrogen o-quinones (19). In the present study, the reaction of lysine with 3,4-EQ has been explored to further our understanding of the nature of the interactions between estrogen quinones and proteins. The results show that lysine reacts by 1,2- and 1,kMichael addition, leading to the formation of two addition products, 4-hydroxyestrone and several estrogen dimeric products.
Experimental Procedures Chemicals. Estrone was purchased from Steraloids (Wilton, NH), and all other reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI). The synthesis of the estrogen oquinone and catechol was carried out as described by Stubenrauch and Knuppen (20). The estrogen o-quinone was also synthesized by oxidation of catechol estrogens by activated MnOz as described previously in our laboratory (21). Caution: The estrogen o-quinone is potentially hazardous and should be handled i n accordance with NIH Guidelines for the Laboratory Use of Chemical Carcinogens (22). Characterization of Compounds. All of the compounds were characterized by NMR, UV, IR, and MS. Melting points (uncorrected) were taken on a Fisher-Johns apparatus. 'HNMR spectra were obtained with a GE 300-MHz NMR spectrometer, and the chemical shift data are reported in parts per million ( 6 ) downfield from tetramethylsilane as an internal standard. The U V spectra were obtained using a Beckman DU70. The IR spectra were obtained on a Nicolet 5DXC FT-IR spectrometer. Mass spectral data were determined on an AEI MS-30, a VG 7070 E-HF, and a Finnigan MAT 95.
0893-228x/94/2707-0696$04.5010 0 1994 American Chemical Society
Chem. Res. Toxicol., Vol. 7, No. 5, 1994 697
Reaction of Lysine with Estrogen Quinone Reaction of 3,4-Estrogeno-Quinone (1) with L-Lysine (Figure 1). L-Lysine (51.47 mg, 0.35 mmol) in 5 mL of water was added in small portions to quinone (1,200 mg, 0.35 mmol) in tetrahydrofuran (10 mL) and was stirred a t room temperature. After addition of lysine, the red-brown-colored solution turned to a dark violet color. The quinone was totally consumed within 5 min (checked by TLC). The reaction solution was diluted with water (15 mL), extracted with ethyl acetate (3 x 50 mL) and ether (2 x 50 mL), dried (over Na~S04),and evaporated. The residues from ethyl acetate and ether were found to be identical (TLC) and were combined. The organic layer indicated over 10 products which were separated using preparative TLC (silica gel GF, 1mm, Analtech, Inc., USA)with the solvent system: 25% EtOAc in benzene. Five fractions which showed steroid structure (checked by lH-NMR) were separated on a silica gel column. Elution of column with 25% EtOAc in benzene yielded product 3, 7.7 mg, Rf = 0.22 in benzene and Rf = 0.86 in 25% EtOAc in benzene. IR (KBr) cm-I 3450,2985,2945, 1742, 1375,1245. lH-NMR (acetone-&) 6 6.58 ( l H , d, 2-H), 6.50 ( l H , d, 1-H), 3.97 (2H, bs, NHz), 2.80 ( l H , S, OH), 0.87 (3H, s, 18-CH3). MS (CI) m / z 285.3 (16.15, MI, 284 (19.88), 85 (29), 73 (30), 61 (100). The parent ion peak is in agreement with ClsHz3NOz. Further separation and purification gave 4-hydroxyestrone (2, 11.0 mg), mp 264-265 "C dec [lit. mp 260-265 "C (20)],Rf = 0.56 in 25% EtOAc in benzene. Further elution of the column gave an orange-red solid (5, 4.1 mg), mp 188-190 "C dec, Rf = 0.47 in 25% EtOAc in benzene. UV (CHZC12)A,, (nm) 228,271,359. IR (KBr) cm-I 3417,3172, 3121, 2954, 2925, 1739, 1734, 1717, 1696, 1653. 'H-NMR (CDC13) 6 7.36 ( l H , S, 2-H), 2.80 (2H, t, CHzNH), 2.65-0.973 (40H, m, methylene, methine, and amino protons), 0.92 (3H, s, 18-CH3). MS (CI) m / z 512.0 (76, M), 496 (17.71, 286 (loo), 149 (22), 69 (30). The parent ion peak is in agreement with CzgHd404. Further separation and purification yielded a pale yellow solid (4, 1.1mg), mp 242-244 "C dec, Rf = 0.40 in 25% EtOAc in benzene. W (CHZC12) A,,= (nm) 216,227. IR (Kl3r) cm-l 3401, 2930, 2900, 2880, 1740, 1490, 1384. 'H-NMR (CDC13) 6 7.29 ( l H , S, OH), 6.71 ( l H , d, J = 8 Hz, 2H), 6.66 ( l H , d, J = 8.0 Hz, 1-H), 4.04 (2H, q, HzNCHz), 2.83 (2H, t, CHzNH), 2.67-1.16 (21H, m, methylene, methine, and amino protons), 0.83 (3H, s, 18-CH3). MS (CI) m l z 371.3 (100, M+ l),259 (12), 124 (12), 110 (ll),74 (25), 61 (11). The parent ion peak is in agreement with C23H34NzOz. The major product from this reaction after separation and purification on column gave a mixture of two dimeric products of 3,4-estrogen o-quinone which were separated using a C18 reverse-phase preparative HPLC (10% MeOH, 90% HzO). Purification on column yielded pure products 6 and 7. Compound 6,an orange-yellow solid, t~ = 4.86 min, 3.2 mg, mp 240244 "C dec, Rf = 0.34 in 25% EtOAc in benzene. W (CHzClz) Amax (nm) 229,466. IR (KBr) cm-' 3440,2962,2926,1740,1735, 1660,1364,1261,1097,1023,902. 'H-NMR (CDC13)6 7.39 ( l H , S, 2'-H), 7.32 ( l H , d, J = 8 Hz, 2-H), 7.22 ( l H , d, J = 8 Hz, 1-H), 5.32 ( l H , bs, OH), 3.00-1.42 (30H, m, methylene and methine protons), 1.22 (3H, s, 18'-CH3), 0.92 (3H, s, 18-CH3). Highresolution E1 MS m / z 568.28 (9.15, MI, 286 (100),269 (311,267 (111, 227 (lo), which is in agreement with C36H4006. Product with t~ = 6.58 min yielded a pale yellow solid (7,4.0 mg), mp 246-248 "C dec, R f = 0.32. UV (CHzClz),A (nm) 231,282. IR (KBr) cm-I 3450, 2930, 2900, 1740, 1735, 1490, 1384, 1299, 1115, 1027. 'H-NMR (CDC13) 6 7.36 ( l H , S, 2'-H), 6.76 ( l H , d, J = 8 Hz, 2-H), 6.67 ( l H , d, J = 8 Hz, 1-HI, 5.28 ( l H , S, OH), 4.95 (2H, s, CHz), 3.06-1.04 (30H, m, methylene and methine protons), 1.19 (3H, s, l8'-CH3), 0.91 (3H, s, 18-CH3). Highresolution E1 MS m l z 581.98 (7.91, M+), 548 (141, 483 (27), 341 (ll),321 (lo), 191 (8.81, 77 (61, which is in agreement with C37H4206.
+
Results and Discussion T h e reaction of 3,4-estrogen o-quinone, 1,with L-lysine in aqueous tetrahydrofuran solution w a s complete within 5 m i n a n d resulted in a very complex mixture of products.
TLC indicated the formation of over 10 reaction products. Six major products were isolated a n d characterized by a combination of various techniques (W,IR, 'H-NMR, MS, HPLC), and their structures are shown in Figure 1. Spectroscopic d a t a (UV, IR, NMR) for compound 2 were found to be identical with those of a n a u t h e n t i c sample of 4-hydroxyestrone (20). T h e formation of 2 m a y b e explained by two different a n d parallel reaction p a t h ways. One pathway involves 1,2-addition of the c-amino group of lysine to the C-3 carbonyl group of t h e quinone, leading to the formation of carbinolamine 8 which undergoes a 1,5-proton shift followed by aromatization as described previously (19). Another m e c h a n i s m for formation of 2 involves a one-electron transfer from t h e amine (reactions 1a n d 2) via a charge-transfer complex, leading to the formation of the amine radical cation a n d the quinone radical anion (23), which c a n abstract a proton (reaction 3) followed by disproportionation t o give t h e catechol (QH2).
RNH,
+ Q t [RNH,-Q]
Q-
+ H+
-+
2 Q H -,QH,
(1)
QH'
(3)
+Q
(4)
One c a n argue that the quinone radical anion (Q-) c a n b e produced by a photochemical reaction involving an excited singlet-state quinone (Q*) (24), as shown in reactions 5 a n d 6.
QYQ* Q*
+Q
Q-
,
+Q+
(5)
(6)
In order t o determine whether the photochemical reaction plays a major role in formation of 2,6,a n d 7, we studied the reaction of 3,4-EQ u n d e r the same solution conditions in t h e absence of lysine. While we were able t o show formation of 2, 6, a n d 7, these compounds were formed only after stirring at room t e m p e r a t u r e for at least 2 h , indicating that the photochemical reaction plays a minor role in formation of t h e reaction products. Thus, our r e s u l t s show that the driving force for formation of 2 involves e i t h e r 1,2addition of the amino group to the C-3 carbonyl group a n d o r oxidation of t h e amino group as shown in reaction 2. The three major products (2,6,7) were isolated by preparative TLC, a n d the mixture of 6 a n d 7 was extracted w i t h methylene chloride a n d separated by HPLC a n d column chromatography. One can explain the formation of t h e dimeric product 6 by several possible p a t h w a y s involving reaction of some of the radical resonance intermediates of 3,4-EQ. Compound 7 was probably formed by the intermolecular cyclization reaction of reduced 6 w i t h CHzCl2 during workup. S u c h a reaction between catechols a n d CHzXz has been reported earlier (25). The s t r u c t u r e of t h e dimeric products 6 a n d 7 w a s deduced essentially from W, MS, a n d NMR d a t a as well as from their acetylated derivatives, 6a a n d 7a. The mass spectrum of 6 showed a molecular ion at 568.28 corresponding t o the molecular m a s s of t h e proposed structure. The products of the oxidative phenolic coupling c a n be formed t h r o u g h a C-C or C - 0 bond (26).
Tabakovic and Abul-Hajj
698 Chem. Res. Toxicol., Vol. 7, No. 5, 1994
&L-lysine
H
O O
OH 2
1
3
COOH
0
I NH2
5
6: R=H 8a: RrOAc
7: R=H
7a: R=OAc
Figure 1. Products from the reaction of 3,4-estroneo-quinone with L-lysine. Confirmation of the presence of a C-0 bond comes from NMR spectra of product 4 showed a singlet at 7.29 ppm the N M R spectrum which shows one singlet at 7.39 ppm (D2O exchanged) corresponding to the hydroxyl group and which can be attributed to one proton of the o-quinone an AB doublet at 6.71 and 6.66 ppm (J = 8.0 Hz). The moiety, and two doublets centered at 7.32 and 7.22 ppm quartet at 4.04 ppm was assigned to the methylene having equal areas and coupling constants (J = 8 Hz) protons on the carbon atom attached to the NH2 group. The triplet at 2.83 ppm was assigned to the methylene which can be attributed to two aromatic protons of the protons on the carbon atom attached to the NH group. catechol moiety. On the basis of NMR spectra, it is not possible to discriminate between four possible isomers Several methylene and methine protons were observed which could arise through a C-0 coupling reaction. between 2.67 and 1.16 ppm and a singlet at 0.83 ppm However, it seems that the structure of 6 shows a which was assigned to the C-18 methyl group. The IR characteristic singlet for the phenolic proton at 5.32 ppm spectrum showed only one carbonyl group due to C-17 which exchanged with DzO. The UV spectrum of 6 (C-01, indicating the absence of the carboxylic carbonyl showed two absorption bands at A- 229 and 466 nm. group. Mass spectra (CI) showed an M+ 1ion at mlz Compound 7 shows a molecular ion at 581.98 in ac371 (100%) corresponding to the proposed structure of 4. cordance with the proposed structure. NMR spectrum Proton NMR spectrum of 5 showed only the presence of 7 shows one singlet at 7.36 ppm and two doublets at 6.76 and 6.67 ppm (J= 8 Hz)as well as a DzO-exchanged of a singlet at 7.36 ppm in the aromatic region which can phenolic proton a t 5.28 ppm and the singlet of the be attributed to the proton at C-2 which shifted to a lower magnetic field due to the presence of the adjacent methylene protons a t 4.95 ppm. The UV spectrum of 7 electron-withdrawing imino group. The IR spectrum showed two absorption bands a t A- 231 and 282 nm. NMR and mass spectral data following acetylation of 6 showed three carbonyl absorption bands at 1739 (C-0 and 7 are consistent with the structures of 6a and 7a. at C-17),1717 ((2-0 carboxylic), and 1696 (C-0 quinone) In the reaction of 1with L-lysine three major products, cm-l, respectively, as well as absorption at 1653 cm-' 3-5 (Figure 11, were isolated and purified using a due to the C-N group. The U V spectrum of 5 showed combination of preparative TLC and column chromatogthree absorption bands at 1,- 228, 271, and 359 nm. raphy. The IR spectrum of 3 showed the characteristic Mass spectra (CI) showed a parent ion at m l z 512 (76%) absorption peaks at 2985 and 2945 cm-l indicating which is in accordance with the proposed structure 5. primary amino group. Proton NMR showed a singlet at Figure 2 shows the pathways for formation of 3-5. It 2.80 ppm (exchanged with DzO) corresponding to the is proposed that 1,Paddition of the a-amino group of hydroxyl group, an AB doublet at 6.58 and 6.50 ppm (J L-lysine takes place at the C-3 carbonyl to give 8. This = 8.1 Hz) corresponding to C-1H and C-2H of the assumption is based on the isolation and identification aromatic ring, and a broad singlet at 3.97 ppm (exof aminophenol3, as well as the fact that the C-3 carbonyl changed with DzO) corresponding to the amino group. group is sterically less hindered. These results are Mass spectra showed a n M+ molecular ion of m l z 285, essentially consistent with those reported earlier for which supports the structure of compound 3. Proton reaction of 3,4-EQ with propylamine (19).Dehydration
+
Reaction of Lysine with Estrogen Quinone
Chem. Res. Toxicol., Vol. 7, No. 5, 1994 699
J
3
COOH
& 0
COOH HC!-NH,
HN
Figure 2. Proposed pathway for the formation of 3,4, and 5. of the intermediate carbinolamine 8 followed by decarboxylation as described earlier (27) gives rise to two resonance anions, 9 and 10. Although one would expect 10 to undergo rearrangement, leading to the formation of the benzoxazole as shown earlier (19, 271, we were unable to isolate the benzoxazole in the present study. While we do not have a definitive reason for the lack of formation of the benzoxazole, it is quite possible that compound 10 rearranges quickly to 11. Protonation of 9 followed by hydrolysis gives rise to 3, while protonation of 10 gives the o-quinoneimine 11. Although several attempts to isolate 11 were not successful, it is proposed as an intermediate based on the isolation and identification of 4 and 5. The o-quinoneimine 11 is presumably reduced by the catechol 2 (QHZ), leading to compound 4. The fate of the intermediate 11 can be explained through its reaction with L-lysine, which proceeds by 1,4-Michael addition, giving rise to intermediate 12,which undergoes oxidation by 3,4-EQ, resulting in the formation of 5. In a previous study on the reaction of 3,4-EQ with propylamine (19), we proposed that the amine adds by 1,6Michael addition, leading to the formation of the C-2 adduct. This assumption was based on the known addition chemistry of thiols to 3,4-EQ (16,28)since NMR data could not differentiate between the C-1 and C-2 protons. However, recent results obtained from calculation of the atomic charges on heavy atoms for 3,4-EQ,3 as well as new studies from addition of soft and hard nucleophiles to 3,4-EQ, clearly show that lysine adds by 1$-Michael addition, leading to the formation of the
lysine adduct a t C-1 as shown in compound 5. Thus, in light of these results the proposed structures in our earlier studies on reaction of propylamine with 3,4-EQ should be revised such that addition is at the C-1 position and not at the C-2 position (19). The formation of catechol estrogens via 2- and 4-hydroxylation of estradiol is now considered a major pathway of estrogen metabolism in humans and animals (29, 30). Further metabolism of catechol estrogens leads to their O-methylated derivatives which are biologically inactive (311, or oxidation to the o-semiquinone and quinones (32)which are strong electrophiles and therefore can bind irreversibly to nucleophilic centers. The recent studies in our labotatory showing that 3,4-EQ can induce DNA damage in MCF-7 cultures (5) and that appropriate chemical modification of estradiol can reduce its carcinogenicity without affecting its estrogenicity (33) are strong evidence that metabolic activation may play a role in the carcinogenicity of estrogens. Covalent DNA binding, which is a characteristic for the majority of chemical carcinogens, probably does not take place with estrogens (34, 35h2 However, strong binding to proteins has been well documented (14, 36, 37) and that the quinonoid moiety may be responsible for their irreversible binding (14, 37). It has been well documented that quinones may be toxic to cells by a number of mecha3 Tabakovic, K., and Abul-Hajj, Y . J. (1994) Estrogen+quinones: Differences in Michael addition of so& and hard nucleophiles. In preparation.
700 Chem. Res. Toxicol., Vol. 7,No. 5, 1994
nisms including redox cycling and the generation of ROS as well as arylation of macromolecules (38).Studies in our laboratory are continuing to investigate the importance of redox cycling in the carcinogenicity of 3,4-EQ ( 5 , 6 ) . Quinones are also known to react with GSH either spontaneously or by catalysis by GSH 5'-transferases (39, 40)leading to its depletion in cells. Once intracellular GSH is depleted, cellular macromolecules, e.g., membrane and other proteins, are alkylated (41).Although we have shown that thiols react with estrogen quinones (15, 161, we believe that the cytotoxicity of 3,4-EQ may be in part due to covalent binding to amine nucleophiles, such as lysine, and not necessarily only due to either depletion of GSH or inhibition of critical thiol groups of proteins. Recent studies in our laboratory have shown that the rate of reaction of 3,4-EQ with the amine nucleophiles is significantly faster than the reaction with thio nucleophile^.^ Detailed investigations on the reaction of 3,4-EQ with model proteins are currently under investigation. In conclusion, we have shown that lysine can react with 3,4-EQ, leading to the formation of a complex mixture of products. These products could be explained through oxidation of the amine by the o-quinone followed by formation of quinone radicals which may be involved in formation of catechols and dimeric products. Furthermore, the a-amino group forms the enamine by 1,2addition followed by 174-Michaeladdition of the €-amino group of lysine. Studies by Trautner and Roberts showed Michael addition first leading to the formation of the aminochrome followed by enamine formation (42). Whether this reversal of reactions is unique to 3,4-EQ remains to be elucidated. The presence of the €-amino group of lysine in proteins could play a significant role not only in the detoxification of estrogen o-quinone leading to the formation of catechols and o-aminophenol, but also may be involved in modification of the protein by forming adducts which may be responsible for the increased genotoxicitylcarcinogenicity of the estrogen o-quinones. Preliminary studies in our laboratory showed that estrogen o-quinones react with bovine serum albumin through the amino group of lysine, leading to the formation of the catechol. Further studies on the role of amino acid side chains in the detoxification andor additions to estrogen o-quinones are currently under investigation.
Acknowledgment. This work was supported by the National Institutes of Health, National Cancer Institute Grant R 0 1 CA57615.
References (1) Kirkman, H. (1984)Estrogen-induced tumors of the kidney in the Syrian hamster. 111. Growth characteristics in the Syrian hamster. Natl. Cancer Znst. Monogr. 1, 1-58. (2) Herbst, A. L., Ulfelder, H., and Poskanzer, D. Z. (1971) Adenocarcinoma of the vagina: Association of maternal stilbesterol therapy with tumor appearance in young women. N . Engl. J.Med. 284,878-881. (3) Bergkvist, L., Adami, H. O., Persson, I., Hoover, R., and Schairer, C. (1989) The risk of breast cancer after estrogen and estrogenprogestin replacement. N . Engl. J. Med. 321, 293-297. (4) Vana, J., Murphy, G. P., Aronoff, B. L., and Baker, H. W. (1979) Survey of primary liver tumors and oral contraceptive use. J . Toxicol. Enuiron. Health 5 , 255-273.
Tabakovic, K., and Abul-Hajj, Y. J. (1994) Estrogen+-quinones: Reaction with side chain mimics of amino acids. In preparation.
Tabakovic and Abul-Hajj (5) Nutter, L. M., Ngo, E. O., and Abul-Hajj, Y. J. (1991) Characterization of DNA damage induced by 3,4-estrone+-quinone in
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Announcements 9th International Conference on Cytochrome P450 The 9th International Conference on Cytochrome P450 will be held July 23-27, 1995, a t the University of Zurich in Switzerland. The meeting will consist of invited lectures, two poster sessions, and two oral presentations of selected posters on the following topics: P450 Topology-ProteinProtein and Protein-Phospholipid Interactions; Reaction Mechanisms and Model Systems; P450 Structure-X-Ray, Sited-Directed Mutagenesis, and Modelling; Heme-Thiolate Proteins Other Than P450; Regulation of P450 Gene Expression; Polymorphism; Electron Transfer and Catalysis; and Plant and Insect P450s. Abstracts for poster sessions must be submitted before May 1, 1995. For further information and abstract submission, please contact: W.-D. Woggon, Chairman, Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Meeting Calendar October 23-27, 1994
Sixth North American ISSX Meeting [Chem. Res. Toxicol. 7 (21, 275, 19941.
November 1-3, 1994
Society for Free Radical Research Satellite Meeting: “Cellular Oxidants: Production and Consequences’’ [Chem. Res. Toxicol. 7 (41, 584, 19941.
November 6- 10, 1994
International Society for Free Radical Research: 7th Biennial Scientific Meeting [Chem. Res. Toxicol. 7 (41, 584, 19941.
January 4-8, 1995
The Fifth International Symposium on Biological Reactive Intermediates [Chem. Res. Toxicol. 7 (21, 276, 19941.
April 22-25, 1995
International ISSX Workshop on Glutathione 5‘-Transferases [Chem.Res. Toxicol. 7 (41, 584, 19941.
