Chem. Res. Toxicol. 1994, 7, 891-896
891
Styrene Metabolism by cDNA-ExpressedHuman Hepatic and Pulmonary Cytochromes P4501 Tamie Nakajima,* Eivor Elovaara,? Frank J. Gonzalez,# Harry V. Gelboin,* Hannu Raunio,g Olavi Pelkonen,O Harri Vainio," and Toshifumi Aoyamal Departments of Hygiene and Biochemistry, Shinshu University School of Medicine, Matsumoto 390, Japan, Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Topeliuksenkatu 41 d, FIN-00250, Helsinki, Finland, National Cancer Institute, Bethesda, Maryland 20892, Department of Pharmacology and Toxicology, University of Oulu, FIN-90220, Oulu, Finland, and International Agency for Research on Cancer, 150 Cours Albert-Thomas, 69372 Lyon, France Received February 7, 1994@ The rate of formation of styrene glycol from styrene was compared in human, rat, and mouse liver microsomes. At a low styrene concentration (0.085 mM), the rates decreased in the order, mouse (2.43 f 0.29 nmol/(mg of proteinamin)) > rat (1.07 f 0.20) > human (0.73 f 0.45); a t a high concentration (1.85 mM), the order was rat (4.21 f 0.72) > mouse (2.72 f 0.11) > human (1.91 f 0.84). Kinetic analysis indicated the presence of a t least two forms of styrenemetabolizing cytochrome P450s with different K, values in human liver microsomes. Styrene was also metabolized in human lung microsomes: the rate of styrene glycol formation was higher in the lung microsomes from smokers than in those from current nonsmokers. The P450 forms responsible for transforming styrene t o styrene glycol were determined by analyzing cDNA-expressed individual P450 forms produced in cultured hepatoma G2 cells by recombinant vaccinia viruses. Of the 12 human P450 forms studied, CYP2B6 and CYP2El existing in human liver a n d o r lungs and CYP2Fl in human lungs were the most active in the forming of styrene glycol, followed by CYPlA2 and CYP2C8. Human CYP3A3, CYP3A4, CYP3A5, and CYP4B1 also catalyzed the metabolism but were much less active. CYP2A6, CYP2C9, and CYP2D6 had only a little detectable activity. CYPlA2, CYP2B6, CYP2C8, CYP2E1, and CYP3AU3A3 were expressed in human liver microsomes, and CYP2C8 was expressed in human lung microsomes, although the expression of CYP2Fl and CYP4B1 could not be investigated. These data indicate that several human hepatic and/or pulmonary P450 forms are capable of metabolizing styrene, albeit at different rates. Styrene is extensively used in a wide range of industrial processes and particularly in the production of plastics and polyester resins ( 1 , 2 ) . Styrene has demonstrated carcinogenic potential in laboratory animals (1), although the potential in humans is still unclear (3-6). Styrene is metabolically converted to styrene-7,8-oxide, which may represent a critical event in the toxicity of styrene (11. A number of P4502 forms can catalyze the oxidation of styrene, as evidenced in rodents (7-91, but only the contribution of CYPBEl(10)to the oxidation has been determined in humans (11).Significant differences exist between man and experimental animals with respect to catalytic activities and the regulation of individual P450 forms (12). Therefore, the specific P450s involved in styrene oxidation should be identified, and their respective roles in various organs and species should be clarified. *To whom correspondence and requests for reprints should be addressed at the Department of Hygiene, Shinshu University School of Medicine, Matsumoto 390,Japan. + Institute of Occupational Health. National Cancer Institute. 5 University of Oulu. 1' International Agency for Research on Cancer. Department of Biochemistry, Shinshu University School of Medicine. Abstract published in Advance ACS Abstracts, October 1, 1994. A part of this work was presented at the International Symposium on Health Hazards of Butadiene and Styrene in Espoo, Finland, 1993. Abbreviations: P450,cytochrome P450;Hep G2,hepatoma G2; DMEM, Dulbecco's modified Eagle medium; PBS, 10 mM sodium phosphate buffer, pH 7.5,containing 0.14 M NaCl; SDS, sodium dodecyl sulfate.
*
@
A method for evaluating the catalytic activities of human P450 enzymes has been developed; it involves vaccinia virus-mediated cDNA expression. This procedure makes it possible t o accurately define the P450 forms that metabolize specific chemicals and carcinogens (13). We investigated the ability of 12 human hepatic and pulmonary P450s to oxidize styrene and their expression in liver and lungs. The ability of microsomes from human liver and lungs to catalyze the oxidation was also examined.
Experimental Procedures Human Liver and Lung Specimens. Liver samples were obtained at laparotomy from 10 patients (eight men and two women) who were undergoing surgery for either primary liver tumors or hepatic metastases at t h e University Hospital in Oulu, Finland. Lung samples were obtained from 38 patients (24 men and 14 women; 27 lung cancer and 11nonlung cancer) undergoing lobectomy or pneumectomy at the Helsinki University Hospital, Finland. A part of the liver sample for immunoblot analysis was obtained at laparotomy from seven patients (men) who were undergoing surgery for hepatic metastases at the Fourth Hospital of Hebei Medical College i n Shijiazhuang, China. The use of the liver and lung tissues was approved by t h e Ethical Committee of t h e Medical Faculty, University of Oulu and Helsinki, and Hebei Medical College, respectively. The smoking habits of the patients were investigated during personal interviews before surgery, and the patients were
0893-228~/94/2707-0891$Q4.5O/Q 0 1994 American Chemical Society
892 Chem. Res. Toxicol., Vol. 7, No. 6, 1994 divided into never smoker, exsmoker who had stopped smoking at least 1 week earlier, and smoker. Animal Liver Specimens. Wistar male rats and B6C3F1 male mice were obtained from Nippon SLC (Shizuoka, Japan). Five rats and five mice were killed at 8 weeks of age for the study on styrene oxidation in the two species. Human, rat, and mouse liver and/or lung microsomal fractions, respectively, were prepared by standard differential centrifugation descrived elsewhere (14). Assay of Styrene Metabolism with Cell. Recombinant vaccinia viruses encoding cDNAs for h u m a n CYPlA2 (15), CYF'2A6 (161,CYP2B6 (171,CYP2C8, and CYP2C9 (181,CYP2D6, and CYP2El (191, CYP2Fl (201, CYP3A3 (211, CYP 3A4 and CYP3A5 (221, CYP4B1 (231, mouse C Y P l A l (24) and CYPlA2 (15),and r a t CYP2Bl (25)and CYP2B2 (261,respectively, were constructed and characterized. Hep G2 cells from the American Type Culture Collection, cultured i n DMEM containing 10% fetal calf serum, were infected with a recombinant virus. Aproximately 160 pmol of P450 was each synthesized i n a 57cm2 dish within 20 h after infection (19). The medium was replaced by DMEM without phenol red, and 50 pL of styrene in 250 mM methanol, giving little inhibition to the reaction, was added to a final concentration of 5 mM to the incubation system. The dish was incubated at 37 "C i n a COZ incubator. Under these conditions, the amount of styrene glycol increased linearly with time u p to 4 h i n any dish. Two hours after the addition of the substrate, an aliquot of 1 mL of the medium was taken, and 0.2 pL of both 15% zinc sulfate and saturated barium hydroxide was added. The mixture was centrifuged at 1800g for 15 min, and 10 pL of the supernatant was taken for the measurement of styrene glycol content i n a high-performance liquid chromatograph with an UV-vis detector and a chromatointegrator. The analytical conditions were as follows: column, 4.0 mm diameter x 250 m m stainless steel column packed with Unisil C18; mobile phase, 10% acetonitrile solution containing 0.25% phosphoric acid at a flow speed of 1.1 m u m i n ; wavelength, 200 nm. The detection limit of styrene glycol was 5 pmol. Styrene-7,8-oxide was not detected in any dish under these conditions. Assay of Styrene Metabolism with Cell Lysate. Hep G2 cells were cultured in DMEM, containing 10% fetal calf serum, and infected with a form of recombinant virus containing human P450. The cells were harvested by scraping from the plates. The cells were washed once with PBS, and cell lysates were prepared by sonication, after which the microsomal fraction was obtained. Styrene metabolism in liver and pulmonary microsomes and i n microsomes from the cell lysate was assessed by measuring the rate of product formation i n a reaction system consisting of a n NADPH-generating system (1 mM NADP, 20 mM glucose 6-phosphate, 2 IU glucose-6-phosphate dehydrogenase, and 50 mM magnesium chloride) and containing 200 p g of liver or cell lysate microsomes or 400 pg of lung microsomal protein, 50 mM potassium phosphate buffer, and 0.037-5.24 mM styrene, to a final volume of 0.5 mL. The reaction was initiated by adding substrate, and the reaction vials were placed in a thermoregulated, shaking water bath (37 "C). After 10 (liver microsomes) or 20 min (lung and lysate microsomes) of incubation, 0.1 mL of a 15% zinc sulfate and saturated barium hydroxide was added to stop the reaction for measurement of product formation. The mixture was centrifuged at l8OOg for 15 min, and 20 pL of the supernatant was injected into t h e high-performance liquid chromatograph under the same conditions as above. The amount of styrene glycol formed increased linearly with respect to incubation time for at least 30 min and u p to 1.0 mg of microsomal protein.
Immunoblotting of Cytochrome P450s in Human Liver and Lungs. For immunoblotting of P450, polyclonal antibodies to P450 were prepared in the laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD: antiCYPlA2, which cross-reacted with CYPlAl; anti-CYP2B1, which cross-reacted with CYP2B2; anti-CYP2C11; anti-CYF'2El; and anti-CYP3A1, which cross-reacted with CYP3A2. Hep G2
Nakajima et al. Table 1. Rates of Metabolism of Styrene [Mean f SD, nmoY(mg of proteinmin)] in Human, Rat, and Mouse Liver Microsomes styrene concentration (mM) species
n
0.085
1.85
humana rat mouse
10
0.73 f 0.45 1.07 f 0.20b 2.43 f 0.29b3C
1.91 f 0.84 4.21 5 0.72b 2.72 & O . l l b , c
5
5
Liver samples were obtained from Finnish donors. b Significant difference (p < 0.05) between human and animals. Significant difference (p < 0.05) between rat and mouse. a
-0
-20
-15
-10
-5
0
5
10
15
2
20
25
Styrene concentration, d-'
Figure 1. Reciprocal of the formation of styrene glycol from styrene in two h u m a n liver microsomes versus reciprocal of styrene concentration. Each point represents the mean of duplicate determinations. cells containing human CYPlA2, CYP2B6, CYP2C8, CYP2E1, and CYP3A4, respectively, were harvested by scraping from the plates. Microsomes of cell lysates were prepared as described above and used as standards for immunostaining of the P450s. Human liver and lung microsomes and the standard samples were separated electrophoretically on 10% SDS-polyacrylamide gel (27). After electrophoretic transfer to 0.45 p m nitrocellulose sheets (Bio-Rad) according to Towbin et al. (281,the sheets were exposed to rabbit anti-CYPs with 1000-fold dilution overnight at 4 "C, followed by exposure to alkaline phosphatase-conjugated goat anti-rabbit IgG (KLP Laboratories, Gaithersburg, MD, USA) and stained using NBTBCIP Substrate Kit (Pierce, Rockford, IL, USA). Statistics. Analysis of variance was performed. When there was any significant difference among the groups, means were tested with Student's t-test.
Results Styrene Oxidation in Human, Rat, and Mouse Liver Microsomes. At a low concentration of styrene, the rate of styrene glycol formation was highest in mouse liver microsomes, followed by those of rat and human (Table 1). At a high styrene concentration, the activity was greatest in rat microsomes, followed by mouse and human microsomes. These results suggest that significant species differences are seen in the activity as reported by Mendrala et al. (29), and analysis of human tissue specimens is of value in estimating styrene metabolism in humans. Kinetics of Styrene Metabolism in Human Liver. The K, and V, values for the formation of styrene glycol were obtained from double-reciprocal plots by means of the least squares method (Figure 1).These data suggest that at least two P450s having different K, values (0.04 and 0.10 mM and 1.21 and 1.28 mM) were responsible for the oxidation of styrene in human liver. The V, associated with the low K , form was 1.05 and 1.39 nmol mg-' min-l and that with the high K, form was 2.53 and 3.38 nmol mg-l min-l. Styrene Metabolism in Human Lungs. The formation of styrene glycol from styrene in human lungs was determined in patients with and without lung cancer and
Human P450s Responsible for Styrene Metabolism
Chem. Res. Toxicol., Vol. 7, No. 6, 1994 893
Table 2. Styrene Metabolism (Mean f SD)in Human Lung Microsome@
patients nonlung cancer lung cancer never smoker exsmoker current smoke+ exsmoker current smoker never smoker exsmoker
+
+
n age 11 46 f 18 27 62 f 7 8 46 f 18 17 63 f 6 11 61 f 8 28 62 f 7 25 57 f 15
styrene glycol (nmol/(mg of protein.20 m i d ) 0.13 f 0.08 0.17 f 0.10 0.12 f 0.06 0.13 f 0.06 0.25 f 0.1Y 0.13 f 0.10 0.18 f 0.06
No patients had medications influencing the activity of cytochrome P450. Pack years of smokers were 37 f 18. Significant difference between current smoker and never smoker and between exsmoker or never smoker plus exsmoker a t p < 0.001. 1A2 8
286
8
9
(0 11
12 13 l4 15
910111213
(6
14 15 b
X
8
1
2
3
4
5
6
7
X8
8 9 10 11 l2
13
(4
P450 286
15b
--
P450 x 9 PO50 2C8
Table 3. Formation of Styrene Glycol from Styrene in Vaccinia Virus-Expressed Human, Mouse, and Rat Cytochrome P450s recombinant virus
cell" (nmoWdish-2h))
lysate6 (nmol/(mg of proteinemin))
wild type CYPlA2 CYP2A6 CYP2B6 CYP2C8 CYP2C9 CYP2D6 CYP2El CYP2F1 CYP3A3 CYP3A4 CYP3A5 CYP4B 1
Human NDC 63.2 f 8.2 2.7 f 0.4 119.6 f 13.4 47.6 f 6.9 5.7 f 0.9 2.7 f 0.4 63.4 f 2.0 103.9 f 4.6 23.6 f 3.8 24.0 f 2.5 11.8 f 1.7 13.3 f 1.0
ND 0.096 ND 0.147 0.050 ND ND 0.161 0.105 0.035 0.034 0.025 0.025
CYPlAl CYPlA2
Mouse 85.0 f 2.7 17.2 f 5.3
0.099 0.031
CYF'2B1 CYF'2B2
Rat 198.8 f 19.0 81.9 f 2.6
0.358 0.092
Hep G2 cells, cultured in dishes containing DMEM and 10% fetal calf serum, were infected with vaccinia virus encoding one of P450 isozymes, and styrene was directly added to the dishes. Each value represents the mean f SD of triplicate determinations. b H e p G2 cells expressing P450 isozyme were destroyed by sonication, and the microsomal fraction was used as a source of enzyme. Each value represents mean of duplicate determinations. ND, not detected.
~ S O e C l f S
baod
2
Ell
_-
.
3
.
4
5
6
7
2€1 8
9
10 11 12 13 14 15 16
3A4/3 1 r
_
_
_
_
2 ;
3
4
5
6
7
3A4,'3 8
9 10 11 12
P450 2E1
13 14 15 b
-
-
POSO 3A413
Figure 2. Immunodetection of cytochrome P450s with polyclonal antibodies, anti-CYPlA2, anti-CYP2B1, anti-CYP2C 11, anti-CYP2El and anti-CYP3Al in human liver and lung microsomes. The left-hand column of each gel contained microsomes including each cytochrome P450 (100 pg of protein) prepared by vaccinia virus-expressed system; columns 1-6 contained human lung microsomes (300 pg of microsomal protein); columns 7- 16 contained human liver microsomes (150 pg).
among never smokers, ex-smokers, and smokers. The only difference observed was that the activity in smokers' lungs was significantly greater than that in the lungs of current nonsmokers (never smokers and ex-smokers) (Table 2). Immunostaining of Human Liver and Lung Cytochrome P450. Anti-CYPlA2 detected a clear band in human liver microsomes but a poor band in lung microsomes: only 2 patients showed a faint band in their lungs (Figure 2). Anti-CYP2B1 detected a clear band in four human liver microsomes and a faint band in six human liver microsomes. However, this antibody did not detect any band at the region of the same molecular weight as that of CYP2B6 in human lungs. Anti-CYP2E1 clearly bound the protein at the region of CYP2E1 in human liver microsomes but slightly in human lung
microsomes. Similarly, anti-CYP 3A1 detected the clear band at the region of CYP3A4/3A3 in human liver microsomes and the slight band in human lung microsomes. Anti-CYP2C1 l detected three bands at the cytochrome P450 region in human liver microsomes and one band in human lung microsomes. Judged from the molecular weight of each protein (18)and a band of synthesized CYP2C8, a band with the highest molecular weight was thought to be CYP2C9; a band with middle molecular weight was CYP2C8; and a band with the lowest molecular weight was unidentifiable. CYP2C8 was expressed in human lungs with one exception, but CYP2C9 was not detected in any lung sample. Styrene Oxidation in Cell System. Styrene glycol could not be formed in Hep G2 cells without cDNA expression of P450 (wild type) (Table 3). Of the 12 human P450 forms studied, CYP2B6 and CYP2F1 were most active in oxidizing styrene; CYPlA2 and CYP2E1 showed similar activity, but lower than that of CYP2B6; CYP2C8 expressed half the activity of CYP2B6; the CYP3A members and CYP4B1 showed a little activity; and the activities of CYP 2A6, CYP2C9, and CYP2D6 were negligible. The Km values for styrene oxidation by CYP2R6 and CYP2E1 obtained from double-reciprocal plots and the least squares method were 0.18 and 0.10 mM, respectively, which were very similar to the low K m values seen in human liver microsomes. Mouse CYPlAl catalyzed the formation of styrene glycol more actively than mouse CYPlA2. It is of interest to note that human and mouse CYPlA2 are orthologous counterparts, but the activity of the former was three times higher than that of the latter. Rat CYP2Bl was the most active of the P450s investigated, and the activity was almost double that of human CYP2B6. The catalytic activity of CYP2B2, which has similar amino acid sequence homology to CYP2B1 (30),
894 Chem. Res. Toxicol., Vol. 7, No. 6, 1994 was less than half that of CYP2B1. Styrene Oxidation in Cell Lysates. In the system of cell lysate, no styrene glycol was detected in the wildtype cells as in the cell system. CYP2El was most active, which finding is inconsistent with the results from the cell system. CYP2B6, CYP2F1, CYPlA2, and CYP2C8 were also active for the oxidation in decreasing order. The activities of CYP3A members and CYP4B1, however, were correspondingly very low. Similarly to the results from the cell system, the activities of the other P450s for the oxidation were negligible. In mouse and rat P450 forms, the activity of each P450 for styrene oxidation in the cell lysate was similar to that in the cell system: the activities by mouse CYPlAl and rat CYP2Bl were greater than those by mouse CYPlA2 and rat CYP 2B2, respectively.
Discussion Styrene is oxidized by cytochrome P450s to electrophilic metabolites (11. Of the 12 human P450 isozymes studied, nine were able to catalyze the oxidation, suggesting that it is difficult to determine the major P450 form responsible for the oxidation in human liver because all of the P450 forms except CYP2Fl and CYP4B1 are expressed in the tissue. Of human liver P450 forms, CYP2B6 and CYP2El were the most effective. Although expression of the human CYP2B6 gene at the enzyme level has not yet been demonstrated (171, a significant amount of protein recognized by anti-CYP2B1was present in the human livers (Figure 2). The K, value for this isozyme was 0.18 mM, being similar to that of the low K, isozyme in human liver. Therefore, CYP2B6 may be important in metabolizing styrene in human liver. AntiCYP2B1-recognized protein was not detected in our lung samples, although the protein is thought to be slightly expressed in lung tissue (311. Guengerich et al. (32) reported that purified human CYP2El catalyzes styrene oxidation at a rate of 2.5 nmol/ (nmol of P450emin). In our study, the average rate of formation of styrene glycol in vaccinia virus-expressed human CYP2El was 63.4 nmol dish-l2 h-’ corresponding to 3.3 nmol/(nmol of P450.min) in the cell system, which agrees well with the observation of Guengerich et al. The K, value for the oxidation by this isozyme was 0.10 mM, which is also similar to that of low K, isozyme in human liver. It remains to be clarified, therefore, which P450 forms are responsible for the higher K, activity measured in human liver. CYP2E1 is strongly expressed in all human livers tested and slightly in human lungs (Figure 2). This form in the liver is induced by the consumption of alcohol (331, suggesting that the drinking habit may influence the rate of styrene metabolism in humans. The ability of each P450 form to oxidize styrene in the cell system was similar to that in the cell lysate, except for CYP2E1, which was the most active of the 12 human P450s investigated in the cell lysate but only the third in the cell culture system. The reason is not known. As for the true activity of styrene oxidation, however, the results from the cell lysate may be more representative. An alternative reaction pathway involving glutathione conjugation with styrene oxide (8) is excluded from the cell lysate but not from the whole Hep G2 cell test system (34). This conclusion is not limited to the activity of CYP2El but applies also to that of all P450 forms. Human CYPlA2 was three times more active than mouse CYPlA2 in metabolizing styrene. This finding
Nakajima et al. suggests that a slight difference in amino acid sequence (24) can significantly affect the activity. Unfortunately, we were unable to investigate the contribution of human CYPlAl to the oxidation. Rat (8)and mouse (Table 3) CYPlAl are more active than rat and mouse CYPlA2. Human CYPlAl may be more heavily involved in the oxidation than human CYPlA2, if the contents of the isozymes in liver are similar to each other. In the CYP2C subfamily, CYP2C8 catalyzed styrene oxidation, but its activity was lower than that of CYPlA2 and CYP2E1; CYP2C9 showed only negligible activity. Although both CYP2C8 and CYP2C9 were expressed in human liver, only CYP2C8 may play a role in the metabolism. In the liver from untreated male rats, CYP2Cll was the major form involved in the oxidation (35). The activity of purified CYP2Cll was double that of rat CYPlAl and triple that of rat CYPlA2 (8). The results suggest that unidentified a CYP2C member(s) with greater activity than CYP2C8 may exist in human liver. CYP3A4 is one of the major forms of P450 in human liver, and it is usually more abundant than CYP3A3 (36). CYP3A5 is expressed polymorphically in the human liver (37). In our human samples, a significant amount of CYP3A413A3 was observed in all livers. Thus, the CYP3A members seem to contribute considerably to the metabolism. Styrene is oxidized, not only by human liver but also by human lungs, although the rate of styrene glycol formation in the lungs is less than 1%of that observed in the liver and only a few percent of that in rat lungs (35). Interestingly, samples from the lungs of current smokers were more active in oxidizing styrene than those from nonsmokers. Human lungs contain CYPlAl (38), which has been suggested to be induced by cigarette smoking (39, 40). CYPlAl may be involved in the lung metabolism of smokers. CYP2Fl and CYP4B1 are thought to be spontaneously expressed in human lungs (20, 231, and the former exhibited a similar level of activity as CYP2B6. It still remains to be clarified which P450 is important in oxidizing styrene in smokers’ a n d or nonsmokers’ lungs. Furthermore, since styrene exposure caused morphological changes in the epithelial cells of the respiratory tract (41), the distribution of these P450 forms in the tissue should be investigated. The activity of styrene oxidation in the liver microsomes from humans in this study was similar to that observed by Mendrala et al. (291, but the activities in rats and mice were quite different. In rat liver, the major P450 involved in styrene oxidation is CYP2C11(35), the expression of which increases with age (42). In our study, the activity in 18-week-old rats was double that of 8-week-old rats.3 Thus, the styrene oxidation in liver microsomes is highly dependent on the level of CYP2C11. The discrepancy between our results and those of Mendrala et al. (29) in the activities in rodents may be explained by the different levels of CYP2C11 in animals used. In conclusion, significant differences in the oxidation of styrene were found among humans, rats, and mice. The activity was always lower in human liver microsomes than in rat or mouse liver microsomes. Of the 12 human P450 forms, nine catalyzed the reaction, suggesting that these forms produce an epoxide intermediate at least in Nakajima, T., Wang, R.-S., Kishi, R., and Katakura, Y. (unpub-
lished data).
Human P450s Responsible for Styrene Metabolism liver and lungs. Since styrene is potentially carcinogenic through electrophilic intermediates, the cytochrome P450 profile in the target organ is important in the development of toxicity.
Acknowledgment. We wish to thank Ms. Ulla Peltonen for her skillful technical assistance in the preparation of human lung microsomes. This work was supported in part by a Grant-in-Aid for General Scientific Research (No. 03454202) from the Ministry of Education, Science and Culture, Japan.
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Announcements 1995 Society of Toxicology Annual Meeting The 1995 Society of Toxicology Annual Meeting will be held March 5-9, 1995, a t the Baltimore Convention Center in Baltimore, Maryland. The annual meetings are the largest toxicology meetings in the world, attracting more than 4500 scientists from 34 countries. The 1995 program includes symposia, workshops, and roundtable discussions, in addition to more than 1600 abstract presentations. Symposia topics include: Toxicant-Induced Alteration of Gene Expression; Carnitine: A Key Toxicological Target?; Second Messengers: Their Role in Immunotoxicity; Selective Protein Covalent Binding and Target Organ Toxicity; Molecular Markers in the Evaluation of Center Chemopreventive Agents; Comparative Interspecies Molecular Toxicology; Mechanisms of Inflammatory Liver Injury: Adhesion Molecules and Cytotoxicity of Neutrophils; Biomarkers for the Study of Toxicity and Human Disease Using the Cell Biology of Entathione; Water Chlorination: Essential Process or Cancer Hazard; Cell Cycle Controls and Chemical Carcinogenesis; Endocrine Modulation of Reproduction; Interactions between Immune and NonImmune Cells in Immunotoxicology; and Use of Gap Junctional Intercellular Communication as a Biomarker for ChemopreventiodChemotherapeutic Drug Development.
For further information, please contact the SOT Headquarters at (7031-438-3115, Fax: (7031438-3113.
Meeting Calendar 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 S-Transferases [Chem.Res. Toxicol. 7 (41, 584, 19941.
July 23-27, 1995
9th International Conference on Cytochrome P450 [Chem.Res. Toxicol. 7 (51, 701, 19943.
