© 1991 S. Karger AG. Basel 0378-7346/91/0321-0004S2.75/0
Gynecol Obstet Invest 1991;32:4-9
Aryl Hydrocarbon Hydroxylase Activity in the First-Trimester Human Placenta: Induction by Carcinogens and Chemoprotectors1 Eytan R. Barnea, SofiAvigdor Fetoplacental Endocrine Unit, Rappaport Institute, Technion, Haifa, Israel
Key Words. Human placenta • Aryl hydrocarbon hydroxylase activity • Xenobiotics Abstract. The activity of aryl hydrocarbon hydroxylase (AHH) was measured in the placenta using radioenzymatic techniques and benzo(a)pyrene (BP) as substrate. The optimal assay conditions were defined, including cofac tor dependence. The Km of BP to the enzyme was 2 pM and Vmax 0.02 nmol/min. When compared to the AHH activity at term (0.35 ± 0.03; mean ± SEM), lst-trimester activity was significantly lower (0.18 ± 0.05; p < 0.05). However, in the rat liver the enzyme activity was even higher than that found at term. In order to determine whether the 1st-trimester placental AHH activity is sensitive to environmental exposures, explants were incubated with two classes of xenobiotics: carcinogens and chemoprotectors. The carcinogen BP at 50 pAf caused a significant (2.7-fold; p < 0.05) increase in the enzyme activity after an incubation period of 6 h. After an incubation period of 24 h, however, no effect was noted. In contrast, 50 \iM 20-methylcholanthrene had no effect. The effect of chemoprotec tors was also examined: at 25 pM, coumarin, 2-(3)-tert-butyl-4-hydroxy-anisole, and l-phenylazo-2-naphthol signif icantly increased 1.7, 1.7-, and 1.8-fold, respectively, the enzyme activity after an incubation period of 24 h (p < 0.05). In conclusion, AHH in the lst-trimester placenta is sensitive to environmental exposures and is induced in vitro by both carcinogen and chemoprotectors.
The metabolism of xenobiotics by the organism is complex [1], In this process, monooxygenase enzyme systems occupy a prominent position which leads to the formation of highly reactive intermediates [2]. Aryl hy drocarbon hydroxylase (AHH) is regarded as a major P450 enzyme complex that is responsible for xenobiotic activation [3-5]. The placenta has long been recognized as having a significant AHH activity [6], At term, this activity is elevated in women who smoked cigarettes during pregnancy [7], Of the methods used to measure the placental AHH activity, the fluorometric method is the most commonly utilized. This assay measures the conversion of ben-
zo(a)pyrene (BP) to its 3- and 9-hydroxy metabolites. Thin-layer and high-pressure liquid chromatography have been also employed to identify products formed by BP metabolism [8-10], In recent years, a sensitive radiometric assay was described which evaluates the overall BP molecule metabolism [11]. In older studies using fluorometric methods [12, 13], the activity of AHH in the 1st trimester human placenta was found to be very low. Whether this low level of activ ity is due to the nature of the assay used in these studies or to the actual low AHH level is currently unresolved.
This work was supported in part by the Israel Cancer Research Fund to E.R.B
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 1:38:53 AM
Introduction
Placental Aryl Hydrocarbon Hydroxylase Activity
Materials and Methods Chemicals (’H)-BP (92 Ci/mmol) was purchased from Amersham (En gland); highly purified unlabellcd BP. 2-(3)-tert-butyl-4-hydroxylanisole (BHA), l-phenylazo-2-naphthol (Sudan I), coumarin, the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), 20-methylcholanthrene (MC), Tris, and cytochrome c were purchased from Sigma (St. Louis, Mo., USA). Placentae After obtaining appropriate consent, a total of 35 placentae were obtained from both elective termination of pregnancy during the 1st trimester (7-12 weeks) and following normal pregnancies at term (38-42 gestational weeks). A history of cigarette smoking and drug use was taken from each patient and recorded. Adult male SpragueDawley rats were sacrificed by decapitation, and their livers were removed. In some cases, the rats were injected intraperitoneally with 20 mg of MC, sacrificed after 3 days, and then their livers were removed. Placental tissues obtained in a sterile manner were im mersed in cold 0.9% NaCl to remove blood. Then they were minced with sharp scissors to remove decidua and membranes. Placental Explant Preparation The lst-trimester explants were prepared as we have previously described [14], Small 50-100-mg fragments were trimmed of decid ua and membranes and then rinsed with 0.9% NaCl. This was fol lowed by rinsing with Dulbecco’s modified Eagle’s medium which contained penicillin 5,000 U/ml, gentamicin 50 pg/ml, and Fungi zone 5,000 U/ml. Incubations were carried out in culture dishes (six replicates per compound or vehicle) which contained 2 ml of media and 1% antibiotic solution with or without 10 or 50 ¡iM BP or MC, and 25 \iM of BHA, Sudan I, or coumarin. Appropriate dishes with vehicle only were used as controls. Incubations were carried out for 6-24 h in an atmosphere of 95% air and 5% CO: at 37 °C. This yielded a pO: of 140-150 mm Hg. In addition, the tissue viability was documented by progressive glucose consumption by the tissue, linear increase in human chorionic gonadotropin secretion in the media, and vital staining with hematoxylin and eosin. The samples were placed on ice after incubation, the media was discarded, and the tissue was further processed. Tissue Processing Placental tissue was homogenized with a Polytron for 1 min with cold 0.25 M sucrose. The homogenate was then centrifuged for 10 min at 4°C at 2,800 rpm. The supernatant was removed and stored at -2 0 °C until assayed within a few days. In some cases, either the whole homogenate or placental microsomes were used.
Microsomes were prepared by centrifugation of the supernatant at 105,000 rpm for 60 min. The pellet was saved and was resuspended in 0.25 M sucrose before assay. The rat liver microsomes were pre pared in the same fashion. They were stored at - 8 0 ° C and were used as an interval standard for each assay. AHH Assay The placental AHH activity was measured using the method of DePierre et al. [11], with some modifications. The incubation mix ture for the AHH assay was placed in widemouthed glass vials. It contained Tris-HCl buffer 50 mM (pH 7.5), MgCL 5 miM, MnCl2 5 \iM, NADPH 1 mM (25 nCi), [3H]-BP, unlabeled BP 20 pA/, and tissue protein in a total volume of 1 ml. Incubates were vortexed and immersed in a water bath for 10 min at 37 °C. At the end of the incubation period, samples were placed on ice, and the reaction was stopped by adding 1 ml of 0.5 MNaOH 80% ethanol solution. Sam ples were extracted by adding 3 ml of hexane and were then shaken vigorously for 5 min, following by centrifugation for 5 min at 2,800 rpm. One milliliter of the lower phase (of the total 2 ml) was removed, and 4 ml of scintillation fluid was added and counted in a Beckman counter with 55% efficiency. Product recovery was 95%. The rat liver microsomes were run as an internal standard. The interassay variability was less than 10%. All samples were run in duplicate. In time studies, protein, substrate, and cofactor depen dence samples were run in triplicate. In preliminary experiments, a NADPH-generating system was used instead of NADPH itself, but it gave less consistent results. We have observed consistent results when both labeled and unlabeled BP were dissolved in acetone. Also, the whole uncentrifuged placen tal homogenate AHH activity was more variable than the centri fuged samples. When the activity in placental microsomes was com pared with that measured in the centrifuged homogenate, the micro somes had a significantly lower AHH activity (data not shown). Therefore, the 2,800-rpm supernatant was used for generating our data. Incubates in the presence of Tris-HCl buffer served as blanks. They had an activity of less than 1% of total counts (table 1). The tissue protein content was measured using the method of Lowry et al. [15] with bovine serum albumin as standard. The AHH activity was expressed as nanomoles product formed per milligram protein per 10 min. Statistical analysis was carried by regression analysis, one-way analysis of variance, and Student’s t test. The results are expressed as mean values ± SEM. p < 0.05 was considered statis tically significant.
Table 1. Counts (cpm) of the AHH assay in five different lsttrimester placentae under standard assay conditions Total
Sample
Blank
Net3
46,912 43,219 45,329 46,987 44,129
766 617 934 1,110 856
411 430 450 380 379
355 183 484 730 477
a
Specific AHH activity.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 1:38:53 AM
In our study, we utilized a sensitive radiometric assay to measure the AHH activity in the placenta using BP as substrate [11], Our aims were to determine the AHH activity of the placenta during the 1st trimester in com parison to term and, moreover, to examine the func tional significance of this enzyme during the 1st trimes ter by studying its response to various xenobiotics in vitro.
5
Barnea/Avigdor
6
Fig. 1. Time-dependent AHH activity in 1st-trimester ( • ) and term (o) placentae. The reaction time was linear for 15 min. Mean values of two different experiments run in triplicate.
Fig. 2. Protein-dependent AHH activity in the 1st-trimester (•) and term (o) placentae. The reaction was linear until 1.2 mg pro tein. Mean values of two different experiments run in triplicate.
c 1.0 - 1 E o ■£ 0.8
Gynecol Obstet Invest 1991;32:4-9
Aryl Hydrocarbon Hydroxylase Activity in the First-Trimester Human Placenta: Induction by Carcinogens and Chemoprotectors1 Eytan R. Barnea, SofiAvigdor Fetoplacental Endocrine Unit, Rappaport Institute, Technion, Haifa, Israel
Key Words. Human placenta • Aryl hydrocarbon hydroxylase activity • Xenobiotics Abstract. The activity of aryl hydrocarbon hydroxylase (AHH) was measured in the placenta using radioenzymatic techniques and benzo(a)pyrene (BP) as substrate. The optimal assay conditions were defined, including cofac tor dependence. The Km of BP to the enzyme was 2 pM and Vmax 0.02 nmol/min. When compared to the AHH activity at term (0.35 ± 0.03; mean ± SEM), lst-trimester activity was significantly lower (0.18 ± 0.05; p < 0.05). However, in the rat liver the enzyme activity was even higher than that found at term. In order to determine whether the 1st-trimester placental AHH activity is sensitive to environmental exposures, explants were incubated with two classes of xenobiotics: carcinogens and chemoprotectors. The carcinogen BP at 50 pAf caused a significant (2.7-fold; p < 0.05) increase in the enzyme activity after an incubation period of 6 h. After an incubation period of 24 h, however, no effect was noted. In contrast, 50 \iM 20-methylcholanthrene had no effect. The effect of chemoprotec tors was also examined: at 25 pM, coumarin, 2-(3)-tert-butyl-4-hydroxy-anisole, and l-phenylazo-2-naphthol signif icantly increased 1.7, 1.7-, and 1.8-fold, respectively, the enzyme activity after an incubation period of 24 h (p < 0.05). In conclusion, AHH in the lst-trimester placenta is sensitive to environmental exposures and is induced in vitro by both carcinogen and chemoprotectors.
The metabolism of xenobiotics by the organism is complex [1], In this process, monooxygenase enzyme systems occupy a prominent position which leads to the formation of highly reactive intermediates [2]. Aryl hy drocarbon hydroxylase (AHH) is regarded as a major P450 enzyme complex that is responsible for xenobiotic activation [3-5]. The placenta has long been recognized as having a significant AHH activity [6], At term, this activity is elevated in women who smoked cigarettes during pregnancy [7], Of the methods used to measure the placental AHH activity, the fluorometric method is the most commonly utilized. This assay measures the conversion of ben-
zo(a)pyrene (BP) to its 3- and 9-hydroxy metabolites. Thin-layer and high-pressure liquid chromatography have been also employed to identify products formed by BP metabolism [8-10], In recent years, a sensitive radiometric assay was described which evaluates the overall BP molecule metabolism [11]. In older studies using fluorometric methods [12, 13], the activity of AHH in the 1st trimester human placenta was found to be very low. Whether this low level of activ ity is due to the nature of the assay used in these studies or to the actual low AHH level is currently unresolved.
This work was supported in part by the Israel Cancer Research Fund to E.R.B
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 1:38:53 AM
Introduction
Placental Aryl Hydrocarbon Hydroxylase Activity
Materials and Methods Chemicals (’H)-BP (92 Ci/mmol) was purchased from Amersham (En gland); highly purified unlabellcd BP. 2-(3)-tert-butyl-4-hydroxylanisole (BHA), l-phenylazo-2-naphthol (Sudan I), coumarin, the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), 20-methylcholanthrene (MC), Tris, and cytochrome c were purchased from Sigma (St. Louis, Mo., USA). Placentae After obtaining appropriate consent, a total of 35 placentae were obtained from both elective termination of pregnancy during the 1st trimester (7-12 weeks) and following normal pregnancies at term (38-42 gestational weeks). A history of cigarette smoking and drug use was taken from each patient and recorded. Adult male SpragueDawley rats were sacrificed by decapitation, and their livers were removed. In some cases, the rats were injected intraperitoneally with 20 mg of MC, sacrificed after 3 days, and then their livers were removed. Placental tissues obtained in a sterile manner were im mersed in cold 0.9% NaCl to remove blood. Then they were minced with sharp scissors to remove decidua and membranes. Placental Explant Preparation The lst-trimester explants were prepared as we have previously described [14], Small 50-100-mg fragments were trimmed of decid ua and membranes and then rinsed with 0.9% NaCl. This was fol lowed by rinsing with Dulbecco’s modified Eagle’s medium which contained penicillin 5,000 U/ml, gentamicin 50 pg/ml, and Fungi zone 5,000 U/ml. Incubations were carried out in culture dishes (six replicates per compound or vehicle) which contained 2 ml of media and 1% antibiotic solution with or without 10 or 50 ¡iM BP or MC, and 25 \iM of BHA, Sudan I, or coumarin. Appropriate dishes with vehicle only were used as controls. Incubations were carried out for 6-24 h in an atmosphere of 95% air and 5% CO: at 37 °C. This yielded a pO: of 140-150 mm Hg. In addition, the tissue viability was documented by progressive glucose consumption by the tissue, linear increase in human chorionic gonadotropin secretion in the media, and vital staining with hematoxylin and eosin. The samples were placed on ice after incubation, the media was discarded, and the tissue was further processed. Tissue Processing Placental tissue was homogenized with a Polytron for 1 min with cold 0.25 M sucrose. The homogenate was then centrifuged for 10 min at 4°C at 2,800 rpm. The supernatant was removed and stored at -2 0 °C until assayed within a few days. In some cases, either the whole homogenate or placental microsomes were used.
Microsomes were prepared by centrifugation of the supernatant at 105,000 rpm for 60 min. The pellet was saved and was resuspended in 0.25 M sucrose before assay. The rat liver microsomes were pre pared in the same fashion. They were stored at - 8 0 ° C and were used as an interval standard for each assay. AHH Assay The placental AHH activity was measured using the method of DePierre et al. [11], with some modifications. The incubation mix ture for the AHH assay was placed in widemouthed glass vials. It contained Tris-HCl buffer 50 mM (pH 7.5), MgCL 5 miM, MnCl2 5 \iM, NADPH 1 mM (25 nCi), [3H]-BP, unlabeled BP 20 pA/, and tissue protein in a total volume of 1 ml. Incubates were vortexed and immersed in a water bath for 10 min at 37 °C. At the end of the incubation period, samples were placed on ice, and the reaction was stopped by adding 1 ml of 0.5 MNaOH 80% ethanol solution. Sam ples were extracted by adding 3 ml of hexane and were then shaken vigorously for 5 min, following by centrifugation for 5 min at 2,800 rpm. One milliliter of the lower phase (of the total 2 ml) was removed, and 4 ml of scintillation fluid was added and counted in a Beckman counter with 55% efficiency. Product recovery was 95%. The rat liver microsomes were run as an internal standard. The interassay variability was less than 10%. All samples were run in duplicate. In time studies, protein, substrate, and cofactor depen dence samples were run in triplicate. In preliminary experiments, a NADPH-generating system was used instead of NADPH itself, but it gave less consistent results. We have observed consistent results when both labeled and unlabeled BP were dissolved in acetone. Also, the whole uncentrifuged placen tal homogenate AHH activity was more variable than the centri fuged samples. When the activity in placental microsomes was com pared with that measured in the centrifuged homogenate, the micro somes had a significantly lower AHH activity (data not shown). Therefore, the 2,800-rpm supernatant was used for generating our data. Incubates in the presence of Tris-HCl buffer served as blanks. They had an activity of less than 1% of total counts (table 1). The tissue protein content was measured using the method of Lowry et al. [15] with bovine serum albumin as standard. The AHH activity was expressed as nanomoles product formed per milligram protein per 10 min. Statistical analysis was carried by regression analysis, one-way analysis of variance, and Student’s t test. The results are expressed as mean values ± SEM. p < 0.05 was considered statis tically significant.
Table 1. Counts (cpm) of the AHH assay in five different lsttrimester placentae under standard assay conditions Total
Sample
Blank
Net3
46,912 43,219 45,329 46,987 44,129
766 617 934 1,110 856
411 430 450 380 379
355 183 484 730 477
a
Specific AHH activity.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 1:38:53 AM
In our study, we utilized a sensitive radiometric assay to measure the AHH activity in the placenta using BP as substrate [11], Our aims were to determine the AHH activity of the placenta during the 1st trimester in com parison to term and, moreover, to examine the func tional significance of this enzyme during the 1st trimes ter by studying its response to various xenobiotics in vitro.
5
Barnea/Avigdor
6
Fig. 1. Time-dependent AHH activity in 1st-trimester ( • ) and term (o) placentae. The reaction time was linear for 15 min. Mean values of two different experiments run in triplicate.
Fig. 2. Protein-dependent AHH activity in the 1st-trimester (•) and term (o) placentae. The reaction was linear until 1.2 mg pro tein. Mean values of two different experiments run in triplicate.
c 1.0 - 1 E o ■£ 0.8
