Reproductive Toxicology, Vol. 6, pp. 363-366, 1992
0890-6238/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
QUINONE REDUCTASE ACTIVITY IN THE FIRST TRIMESTER PLACENTA: EFFECT OF CIGARETTE SMOKING AND POLYCYCLIC AROMATIC HYDROCARBONS S. AVIGDOR,* D. ZAKHEIM,* a n d E. R. BARNEA*t *Feto-Placental Endocrine Unit, Rappaport Institute, Technion, Haifa, Israel; tThe University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical Center at Camden, Cooper Hospital University Medical Center, Department Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Camden, New Jersey Abstract - - Quinone reductase (QR) is considered a major protective enzyme against cancer in the organism. In this study, the activity of QR was measured in first trimester placental tissue using colorimetrictechniques. There were no significant differences between the mean enzyme activity of women who smoked more than 20 cigarettes per day during pregnancy and of nonsmokers (0.50 +_0.09 compared with 0.51 + 0.15 ~mol/mg protein/10 min, respectively). Among the polycyclicaromatic hydrocarbons (PAH) studied, dimethyl benzanthracene (DMBA) increased QR activity in a dose-dependent manner in the first trimester placental explants at the 10- to 100-~M range after 6 h of incubation (440% increase) with the highest concentration.The effect of other PAH of different potency added at 50-~tM concentrations showed that benz(a)anthracene (BA), dibenzo(a,h)anthracene (DBHA), dibenzo(a,c)anthracene (DBCA), or chrysene (CH) caused a significant 2- to 3-fold increase in the enzymatic activity after 6 h of incubation. At 24 h 50-~tM DBCA effect was also stimulatory, while the 10-~M DMBA effect almost reached statistical significance. However, no differences were encountered in the response of placental tissues to PAH between cigarette smokers and nonsmokers at 6 and 24 h. The present data indicate that placental QR activity is increased by exposure to PAH in vitro, but it does not appear to be affected by in vivo exposure to cigarette smoking. Thus, the early placenta appears to have a significantpotential to inactivate carcinogens/mutagens locally, thereby limiting their transfer to the embryo. Key Words: quinonereductase;enzyme;cigarettesmoke;carcinogen.
INTRODUCTION
cent evidence that in first trimester placental explants, the monooxygenase (EH) is induced in vitro by various xenobiotics, and this induction is coordinated with that of COMT; AHH activity is also inducible by xenobiotics (I0, 11). Quinone reductase (QR) is considered an important enzyme that protects against cancer and environmental toxicity (1213). We have recently reported that mercury, a toxic metal, induces this enzyme activity in term placental explants (14). In the present study we have examined the effect of maternal cigarette smoking on basal QR activity in the first trimester placenta, as well as the effect of various PAH, a principal component of smoke, on explant enzyme activity. Our data show that QR activity is not modified by maternal smoking; however, the enzyme activity is increased by short-term exposure to PAH in vitro.
Following implantation, trophoblastic cells serve as support and a selective barrier for the developing embryo. However, the information on the metabolic competence of the placenta is based primarily on observations made at term. Where it was shown that the placenta is capable of activating xenobiotics, thereby forming reactive compounds that may harm the conceptus through hydroxylation processes, principally by aryl hydrocarbon hydroxylase (AHH) as well as estrogen hydroxylase (EH) (1-4), the placenta also has protective enzymes whose activities are modified in high-risk conditions. Among them, the activities of gluthatione S-transferase (GST), catechol-O-methyl transferase (COMT), and monoamine oxidase (MAO) have been widely investigated (5-9). In contrast, the information on xenobiotic metabolism in the first trimester is marginal. We have presented re-
MATERIALS AND M E T H O D S Chemicals
Addresscorrespondenceto EytanR. Barnea,M,D.,7447 Old York Road, MelrosePark, PA 19126. Received 3 Decenber 1990;Final revision received2 December 1991;Accepted 22 December 1991.
Dimethyl benz(a)anthracene (DMBA), benz(a)anthracene (BA), dibenzo(a,h)anthracene (DBHA), dibenzo(a,c)anthracene (DBCA), chrysene (CH), 2,6363
364
Reproductive Toxicology
Volume 6, Number 4, 1992
dichlorophenol, dicoumarol, and reduced nicotine adenine dinucleotide (NADA) or NADPH were purchased from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM) was purchased from Beit Haemek (Israel).
800 × g at 4 °C for 5 rain. The supernatant obtained was used for all assays. After incubation, the explants were removed from surrounding media and homogenized with 1.5 mL of cold 0.25 M sucrose in a manner similar to that used for fresh tissue.
Collection of placentae
QR assay
After obtaining appropriate consent, 37 first trimester placentae (8-11) were collected from elective pregnancy terminations using suction curretage. Placentae were obtained from 12 women who smoked more than 20 cigarettes per day until the day of the procedure but were otherwise healthy. The remainder were 25 women who were healthy nonsmokers. Neither smokers nor nonsmokers used any medications. The placentae were rinsed in cold 0.9% NaC1 until free of blood.
Placental QR activity was measured according to the method reported previously by Ernster (18) and recently reported by us in the term placenta (14). Briefly, the assay mixture in a total volume of 3 mL contained 25 mM Tris-HC1 buffer (pH 7.4), 80 uM 2,6-dichlorophenolindophenol, 20 gM NADH or NADPH, and placental supernatant (0.1 to 0.5 mg protein). The samples were incubated for 10 min at 25 °C. The rate of reduction of 2,6-dichlorophenolindophenol was measured at 600 nm by a Beckman spectrophotometer. The reaction was linear for the range of 1 to 10 min, at 0.1 to 0.8 mg protein concentration and substrate, which was added in excess. The reaction was inhibited more than 90% by additional 1 mM dicoumarol. No differences in the QR activity were noted between incubations made with NADH or NADPH.
Explant preparation The method for preparation of placental explants was previously reported (9,15). Briefly, tissue was separated from membranes and decidua, using a dissecting microscope, and washed in DMEM plus 1% antibiotics (penicillin 10,000 units/mL, streptomycin 10 #g/mL, and amphotericin B 25 #g/mL). Fifty-mg wet weight explants ( 1 to 3 mg protein) were prepared and incubated in 2 mL of DMEM plus 1% antibiotics with the various PAH compounds tested dissolved in acetone, or with acetone alone (vehicle control) for 6 or 24 h in an atmosphere of 95% air and 5% CO2. Each treatment group or vehicle-treated explant group was run in six replicates per placenta. At the end of the incubation period, the explants were removed and stored at -20 °C until assayed within a few days. Each explant's viability was established by the linear increase in hCG secretion during culture, progressive glucose consumption, and by vital staining with hematoxylin-eosin. In addition, we have examined in detail the viability ofexplants following exposure to carcinogens (16), finding that compared to 6 h, incubation with 50 #M BP for 24 h caused a more pronounced increase in hCG secretion in the media. In addition, following these exposures at 24 h, spontaneous hCG pulsatility in superfusion was increased compared to 6 h. Finally, at both time points, hCG was increased by pulses of G n R H analog given during superfusion. We have previously reported that this analog stimulates hCG secretion in superfusion (17). Together these data point to the maintenance of placental viability following exposure to carcinogens.
Homogenate preparation Collected placentae were homogenized with 4 volumes of cold 0.25 M sucrose and centrifuged at
Protein estimation Tissue protein content was measured using the method of Lowry and colleagues (19).
Statistical analysis Statistical analysis was carried out by using Student's t test and one-way ANOVA. P values
0890-6238/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
QUINONE REDUCTASE ACTIVITY IN THE FIRST TRIMESTER PLACENTA: EFFECT OF CIGARETTE SMOKING AND POLYCYCLIC AROMATIC HYDROCARBONS S. AVIGDOR,* D. ZAKHEIM,* a n d E. R. BARNEA*t *Feto-Placental Endocrine Unit, Rappaport Institute, Technion, Haifa, Israel; tThe University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical Center at Camden, Cooper Hospital University Medical Center, Department Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Camden, New Jersey Abstract - - Quinone reductase (QR) is considered a major protective enzyme against cancer in the organism. In this study, the activity of QR was measured in first trimester placental tissue using colorimetrictechniques. There were no significant differences between the mean enzyme activity of women who smoked more than 20 cigarettes per day during pregnancy and of nonsmokers (0.50 +_0.09 compared with 0.51 + 0.15 ~mol/mg protein/10 min, respectively). Among the polycyclicaromatic hydrocarbons (PAH) studied, dimethyl benzanthracene (DMBA) increased QR activity in a dose-dependent manner in the first trimester placental explants at the 10- to 100-~M range after 6 h of incubation (440% increase) with the highest concentration.The effect of other PAH of different potency added at 50-~tM concentrations showed that benz(a)anthracene (BA), dibenzo(a,h)anthracene (DBHA), dibenzo(a,c)anthracene (DBCA), or chrysene (CH) caused a significant 2- to 3-fold increase in the enzymatic activity after 6 h of incubation. At 24 h 50-~tM DBCA effect was also stimulatory, while the 10-~M DMBA effect almost reached statistical significance. However, no differences were encountered in the response of placental tissues to PAH between cigarette smokers and nonsmokers at 6 and 24 h. The present data indicate that placental QR activity is increased by exposure to PAH in vitro, but it does not appear to be affected by in vivo exposure to cigarette smoking. Thus, the early placenta appears to have a significantpotential to inactivate carcinogens/mutagens locally, thereby limiting their transfer to the embryo. Key Words: quinonereductase;enzyme;cigarettesmoke;carcinogen.
INTRODUCTION
cent evidence that in first trimester placental explants, the monooxygenase (EH) is induced in vitro by various xenobiotics, and this induction is coordinated with that of COMT; AHH activity is also inducible by xenobiotics (I0, 11). Quinone reductase (QR) is considered an important enzyme that protects against cancer and environmental toxicity (1213). We have recently reported that mercury, a toxic metal, induces this enzyme activity in term placental explants (14). In the present study we have examined the effect of maternal cigarette smoking on basal QR activity in the first trimester placenta, as well as the effect of various PAH, a principal component of smoke, on explant enzyme activity. Our data show that QR activity is not modified by maternal smoking; however, the enzyme activity is increased by short-term exposure to PAH in vitro.
Following implantation, trophoblastic cells serve as support and a selective barrier for the developing embryo. However, the information on the metabolic competence of the placenta is based primarily on observations made at term. Where it was shown that the placenta is capable of activating xenobiotics, thereby forming reactive compounds that may harm the conceptus through hydroxylation processes, principally by aryl hydrocarbon hydroxylase (AHH) as well as estrogen hydroxylase (EH) (1-4), the placenta also has protective enzymes whose activities are modified in high-risk conditions. Among them, the activities of gluthatione S-transferase (GST), catechol-O-methyl transferase (COMT), and monoamine oxidase (MAO) have been widely investigated (5-9). In contrast, the information on xenobiotic metabolism in the first trimester is marginal. We have presented re-
MATERIALS AND M E T H O D S Chemicals
Addresscorrespondenceto EytanR. Barnea,M,D.,7447 Old York Road, MelrosePark, PA 19126. Received 3 Decenber 1990;Final revision received2 December 1991;Accepted 22 December 1991.
Dimethyl benz(a)anthracene (DMBA), benz(a)anthracene (BA), dibenzo(a,h)anthracene (DBHA), dibenzo(a,c)anthracene (DBCA), chrysene (CH), 2,6363
364
Reproductive Toxicology
Volume 6, Number 4, 1992
dichlorophenol, dicoumarol, and reduced nicotine adenine dinucleotide (NADA) or NADPH were purchased from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM) was purchased from Beit Haemek (Israel).
800 × g at 4 °C for 5 rain. The supernatant obtained was used for all assays. After incubation, the explants were removed from surrounding media and homogenized with 1.5 mL of cold 0.25 M sucrose in a manner similar to that used for fresh tissue.
Collection of placentae
QR assay
After obtaining appropriate consent, 37 first trimester placentae (8-11) were collected from elective pregnancy terminations using suction curretage. Placentae were obtained from 12 women who smoked more than 20 cigarettes per day until the day of the procedure but were otherwise healthy. The remainder were 25 women who were healthy nonsmokers. Neither smokers nor nonsmokers used any medications. The placentae were rinsed in cold 0.9% NaC1 until free of blood.
Placental QR activity was measured according to the method reported previously by Ernster (18) and recently reported by us in the term placenta (14). Briefly, the assay mixture in a total volume of 3 mL contained 25 mM Tris-HC1 buffer (pH 7.4), 80 uM 2,6-dichlorophenolindophenol, 20 gM NADH or NADPH, and placental supernatant (0.1 to 0.5 mg protein). The samples were incubated for 10 min at 25 °C. The rate of reduction of 2,6-dichlorophenolindophenol was measured at 600 nm by a Beckman spectrophotometer. The reaction was linear for the range of 1 to 10 min, at 0.1 to 0.8 mg protein concentration and substrate, which was added in excess. The reaction was inhibited more than 90% by additional 1 mM dicoumarol. No differences in the QR activity were noted between incubations made with NADH or NADPH.
Explant preparation The method for preparation of placental explants was previously reported (9,15). Briefly, tissue was separated from membranes and decidua, using a dissecting microscope, and washed in DMEM plus 1% antibiotics (penicillin 10,000 units/mL, streptomycin 10 #g/mL, and amphotericin B 25 #g/mL). Fifty-mg wet weight explants ( 1 to 3 mg protein) were prepared and incubated in 2 mL of DMEM plus 1% antibiotics with the various PAH compounds tested dissolved in acetone, or with acetone alone (vehicle control) for 6 or 24 h in an atmosphere of 95% air and 5% CO2. Each treatment group or vehicle-treated explant group was run in six replicates per placenta. At the end of the incubation period, the explants were removed and stored at -20 °C until assayed within a few days. Each explant's viability was established by the linear increase in hCG secretion during culture, progressive glucose consumption, and by vital staining with hematoxylin-eosin. In addition, we have examined in detail the viability ofexplants following exposure to carcinogens (16), finding that compared to 6 h, incubation with 50 #M BP for 24 h caused a more pronounced increase in hCG secretion in the media. In addition, following these exposures at 24 h, spontaneous hCG pulsatility in superfusion was increased compared to 6 h. Finally, at both time points, hCG was increased by pulses of G n R H analog given during superfusion. We have previously reported that this analog stimulates hCG secretion in superfusion (17). Together these data point to the maintenance of placental viability following exposure to carcinogens.
Homogenate preparation Collected placentae were homogenized with 4 volumes of cold 0.25 M sucrose and centrifuged at
Protein estimation Tissue protein content was measured using the method of Lowry and colleagues (19).
Statistical analysis Statistical analysis was carried out by using Student's t test and one-way ANOVA. P values
