Cigarette Smoke Inhibits Cytosolic Hydrolase of Human Lung*
but not Microsomal
Epoxide
Stefano Petruzzelli, Marina Franchi, Luca Gronchi, Alberto Janni, Franz Oesch, Gian Maria Pacifici & Carlo Giuntini Respiratory Pathophysiology Unit, 2nd Medical Clinic, Section of Thoracic Surgery, and Department of Biomedicine, University of Pisa, Italy and Institute of Toxicology, University of Mainz, Germany
cigarette smoke exposure on the activity of cytosolic and microsomal epoxide hydrolase (EH) has been investigated in human lung. Patients were classified as ’recent smokers’ (n 9) or ’non-recent smokers’ (n 10) according to whether they were or were not still smoking 1 month before surgery. Cytosolic EH was measured with
The effect of
=
=
H] 3 [ trans-
stilbene oxide as a substrate, whereas microsomal EH was measured with [7H]styrene 3 oxide as a substrate. Microsomal EH activity did not differ between recent smokers (2.51 -1mg -1 mg ± 0.93 nmol min ), -1 ) and non-recent smokers (2.74 ± 1.10 nmol min -1 whereas cytosolic EH activity was significantly lower in recent smokers (6.46 ± 1.79 pmol -1 -1 -1) min mg P < 0.05). , mg -1 than in non-recent smokers (8.41 ± 2.09 pmol min with the number of that had was correlated EH days passed since the activity Cytosolic cessation of smoking (r 0.58, P < 0.05) and the effect was dose-dependent, since the enzyme activity was inversely correlated with the number of cigarettes smoked per day (r 0.85, P < 0.01). This suggests that recent smoking exposure inhibits the activity of cytosolic EH but not microsomal EH, and that the inhibition increases with the number of cigarettes smoked per day. The contribution of cytosolic enzymes to xenobiotic metabolism may be remarkable in extrahepatic tissues. The inhibition of cytosolic EH by tobacco smoke may reduce the inactivation of carcinogenic epoxides in human lung tissues and so may increase =
=
a
person’s susceptibility
to
lung cancer.
Introduction smoke has been associated with an increased incidence of cancer in the human respiratory tract. Smoke components, such as the polynuclear aromatic hydrocarbons (PAH), require metabolic activation into epoxides before exerting their carcinogenic activity.2 Parent PAH are first transformed by cytochrome P450 dependent monooxygenases into epoxides, which may be further metabolized by (i) epoxide
Cigarette
hydrolase (EH) to dihydrodiols, (ii) conjugated to glutathione by the glutathione S-transferase (GST), or (iii) rearranged non-enzymatically to phenols. 3,4 EH may play a dual role in the fate of dihydrodiols, since these intermediate metabolites may be inactivated by conjugation with the glucuronyl transferase,s or they may be the substrate for a second monooxygenation, with *
This work has been supported Petruzzelli.
by the
C.N.R. of
the formation of diol-epoxides, which are highly mutagenic and carcinogenic.&dquo; EH activity has been found to be widely distributed in the respiratory tractl° and in different cellular
compartments.11
In particular, microsomal and cytosolic EH activities may vary with species, and tissues, and also the immunological properties, subcellular distribution, sensitivity to inducers and inhibitors may be different for the two enzymes.ll-13 We have previously demonstrated that recent exposure to cigarette smoke (within the preceding 30 d) may differentially affect the activity of certain drug-metabolizing enzymes in human lung tissue, resulting in the induction of microsomal EH and inhibition of GST.l4,is Since cytosolic enzymes may contribute more than the microsomal enzymes to xenobiotic metabolism
Italy Special Project "Oncology",
contract no. 88.00695.44.
Correspondence: S.
Downloaded from het.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015
100
in extrahepatic tissues, 16 the transient imbalance between activating and inactivating enzymes may influence cell susceptibility to the effect of carcinogens.4 This prompted us to ascertain whether cigarette smoke had comparable or different effects on the activity of microsomal and cytosolic EH activity in human lung tissue.
method of Jerina et al., 17 using styrene-7,8-oxide as substrate. Cytosolic EH activity was measured as described by Mertes et al. ,18 using transstilbene oxide as substrate. The protein content of both the microsomal and cytosolic fractions was determined by the method of Lowry et a1.19
Materials and methods
Results
Chemicals Radioactive [7-’Hlstyrene-7,8-oxide (specific activity 99.6 mCi mmol-1) was obtained from Amersham International (Amersham, England). (3H]trans-stilbene oxide (specific activity 13.8 mCi mmol-1) was synthetized in the Institute of Toxicology, University of Mainz (Mainz, Germany). Radioactive styrene oxide and transstilbene oxide were purified by TLC, the final radiochemical purity being greater than 99%. Unlabelled styrene oxide and trans-stilbene oxide and trans-stilbene glycol were obtained
sex, smoking habit and the diagnosis of each patient, as well as the activities of cytosolic and microsomal EH, are summarized in Table 1. The ages ranged from 27 to 74 years (mean ± s.d. 60 ± 10 years). Seventeen patients had lung cancer and only two had non-malignant pulmonary diseases (bullous emphysema and lung abscess, respectively). According to the study criteria, nine patients were defined as recent smokers and ten as non-recent smokers; five of the latter had stopped smoking 2-10 years before surgery. The cumulative smoke exposure of patients, expressed as the number of pack-years, ranged from 15 to 100 pack-years (mean ± s.d. 58 ± 22). The protein content and the microsomal and the cytosolic EH activities, correlated neither with the age of the lung donor nor with the number of pack-years. Recent smokers and non-recent smokers did not differ with respect to age (57 ± 13 vs 64 ± 6 years) or pack-years (59 ± 25 vs 57 ± 21). There was no difference either in the microsomal (16.72 ± 3.33 mg g-1 tissue vs 15.67 ± 2.19 mg g tissue) and cytosolic (21.03 ± 2.20 mg g tissue vs 20.33 ± 1.70 mg g tissue) protein content. Microsomal and cytosolic EH activities were not significantly correlated each other either in the entire group of patients (r = 0.45) or in recent smokers and non-recent smokers separately (r 0.28 and r = 0.57, EH activity did not Microsomal respectively). differ between recent smokers (2.51 ± 0.93 nmol min 1 mg 1) and non-recent smokers (2.74 ± 1.10 nmol min-l mg-1). In contrast, cytosolic EH lower in recent smokers activity was (6.46 ± 1.79 pmol min mg-1) than in non-recent smokers (8.41 ± 2.09 pmol min mg-1, P < 0.05). This may suggest that recent smoking exposure selectively inhibits the activity of the cytosolic but not the microsomal EH. We searched for a possible relationship between exposure to tobacco smoke, expressed as the period since smoking ceased and the number of cigarettes smoked per day, with the activity of cytosolic EH. Cytosolic EH was correlated with the number of days that had elapsed since the smoking was stopped (r = 0.58, P < 0.05; Figure 1), as investigated up to 6
from Aldrich
(Milano, Italy).
Patients Nineteen patients submitted to open chest surgery for lung cancer or other non-neoplastic pulmonary diseases were included in the study. A standardized questionnaire was administered to each patient, in order to collect personal data with special regard to smoking habits. Patients were classified as ’recent smokers’ if they had smoked within the last month before surgery, and ’non-recent smokers’ were those who had stopped smoking more than 1 month before their
operations. Specimens of peripheral lung tissue were sampled at surgery. From lung cancer patients, tissue specimens were sampled from a normal portion of tissue as far away as possible from the tumor. Lung specimens were immediately frozen and were stored at -80°C until assayed. Subcellular isolation and assays
Aliquots (2.5 g) of lung tissue were minced and homogenized in five volumes of 0.25 M sucrose with a Polytron (Kinematica, Luzern, Switzerland). The homogenates were filtered on gauze, rehomogenized in a Potter-Elvehjem apparatus and then centrifuged at 9000 g for 15 min. The supernatant was then centrifuged at 105 000 g for 1 h. The supernatants were investigated as the cytosolic fractions. The resulting pellets were resuspended in 0.1 M Tris-HCl buffer (pH 7.4) containing 30% glycerol and studied as the microsomal fraction. Both microsomal and cytosolic fractions were stored at -80°C. Microsomal EH activity was determined according to the
Age,
Downloaded from het.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015
=
significantly
101
Table 1 surgery.
Age,
sex,
smoking habit, diagnosis
and
epoxide hydrolase activity
in 19
patients
submitted to chest
Note. ’Recent smoker’ was defined as someone who was currently smoking or who had refrained from smoking for less than 30 d; ’non-recent smoker’ comprised those who had never smoked or who stopped it more than 30 days previously. EH cyt. cytosolic epoxide hydrolase; EH mic microsomal epoxide hydrolase =
=
Figure 1 Correlation between cytosolic epoxide hydrolase activity (ordinate) and the number of days of refraining from smoking (abscissa) in lung tissue from
Figure
smokers.
recent smokers.
months from surgery, with the lowest activity in patients who had been smoking on the day before surgery. Furthermore in recent smokers the inhibitory effect was dose-dependent, since the enzyme activity was inversely correlated with the number of cigarettes smoked per day (r = 0.85, P
some
but not Microsomal
Epoxide
Stefano Petruzzelli, Marina Franchi, Luca Gronchi, Alberto Janni, Franz Oesch, Gian Maria Pacifici & Carlo Giuntini Respiratory Pathophysiology Unit, 2nd Medical Clinic, Section of Thoracic Surgery, and Department of Biomedicine, University of Pisa, Italy and Institute of Toxicology, University of Mainz, Germany
cigarette smoke exposure on the activity of cytosolic and microsomal epoxide hydrolase (EH) has been investigated in human lung. Patients were classified as ’recent smokers’ (n 9) or ’non-recent smokers’ (n 10) according to whether they were or were not still smoking 1 month before surgery. Cytosolic EH was measured with
The effect of
=
=
H] 3 [ trans-
stilbene oxide as a substrate, whereas microsomal EH was measured with [7H]styrene 3 oxide as a substrate. Microsomal EH activity did not differ between recent smokers (2.51 -1mg -1 mg ± 0.93 nmol min ), -1 ) and non-recent smokers (2.74 ± 1.10 nmol min -1 whereas cytosolic EH activity was significantly lower in recent smokers (6.46 ± 1.79 pmol -1 -1 -1) min mg P < 0.05). , mg -1 than in non-recent smokers (8.41 ± 2.09 pmol min with the number of that had was correlated EH days passed since the activity Cytosolic cessation of smoking (r 0.58, P < 0.05) and the effect was dose-dependent, since the enzyme activity was inversely correlated with the number of cigarettes smoked per day (r 0.85, P < 0.01). This suggests that recent smoking exposure inhibits the activity of cytosolic EH but not microsomal EH, and that the inhibition increases with the number of cigarettes smoked per day. The contribution of cytosolic enzymes to xenobiotic metabolism may be remarkable in extrahepatic tissues. The inhibition of cytosolic EH by tobacco smoke may reduce the inactivation of carcinogenic epoxides in human lung tissues and so may increase =
=
a
person’s susceptibility
to
lung cancer.
Introduction smoke has been associated with an increased incidence of cancer in the human respiratory tract. Smoke components, such as the polynuclear aromatic hydrocarbons (PAH), require metabolic activation into epoxides before exerting their carcinogenic activity.2 Parent PAH are first transformed by cytochrome P450 dependent monooxygenases into epoxides, which may be further metabolized by (i) epoxide
Cigarette
hydrolase (EH) to dihydrodiols, (ii) conjugated to glutathione by the glutathione S-transferase (GST), or (iii) rearranged non-enzymatically to phenols. 3,4 EH may play a dual role in the fate of dihydrodiols, since these intermediate metabolites may be inactivated by conjugation with the glucuronyl transferase,s or they may be the substrate for a second monooxygenation, with *
This work has been supported Petruzzelli.
by the
C.N.R. of
the formation of diol-epoxides, which are highly mutagenic and carcinogenic.&dquo; EH activity has been found to be widely distributed in the respiratory tractl° and in different cellular
compartments.11
In particular, microsomal and cytosolic EH activities may vary with species, and tissues, and also the immunological properties, subcellular distribution, sensitivity to inducers and inhibitors may be different for the two enzymes.ll-13 We have previously demonstrated that recent exposure to cigarette smoke (within the preceding 30 d) may differentially affect the activity of certain drug-metabolizing enzymes in human lung tissue, resulting in the induction of microsomal EH and inhibition of GST.l4,is Since cytosolic enzymes may contribute more than the microsomal enzymes to xenobiotic metabolism
Italy Special Project "Oncology",
contract no. 88.00695.44.
Correspondence: S.
Downloaded from het.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015
100
in extrahepatic tissues, 16 the transient imbalance between activating and inactivating enzymes may influence cell susceptibility to the effect of carcinogens.4 This prompted us to ascertain whether cigarette smoke had comparable or different effects on the activity of microsomal and cytosolic EH activity in human lung tissue.
method of Jerina et al., 17 using styrene-7,8-oxide as substrate. Cytosolic EH activity was measured as described by Mertes et al. ,18 using transstilbene oxide as substrate. The protein content of both the microsomal and cytosolic fractions was determined by the method of Lowry et a1.19
Materials and methods
Results
Chemicals Radioactive [7-’Hlstyrene-7,8-oxide (specific activity 99.6 mCi mmol-1) was obtained from Amersham International (Amersham, England). (3H]trans-stilbene oxide (specific activity 13.8 mCi mmol-1) was synthetized in the Institute of Toxicology, University of Mainz (Mainz, Germany). Radioactive styrene oxide and transstilbene oxide were purified by TLC, the final radiochemical purity being greater than 99%. Unlabelled styrene oxide and trans-stilbene oxide and trans-stilbene glycol were obtained
sex, smoking habit and the diagnosis of each patient, as well as the activities of cytosolic and microsomal EH, are summarized in Table 1. The ages ranged from 27 to 74 years (mean ± s.d. 60 ± 10 years). Seventeen patients had lung cancer and only two had non-malignant pulmonary diseases (bullous emphysema and lung abscess, respectively). According to the study criteria, nine patients were defined as recent smokers and ten as non-recent smokers; five of the latter had stopped smoking 2-10 years before surgery. The cumulative smoke exposure of patients, expressed as the number of pack-years, ranged from 15 to 100 pack-years (mean ± s.d. 58 ± 22). The protein content and the microsomal and the cytosolic EH activities, correlated neither with the age of the lung donor nor with the number of pack-years. Recent smokers and non-recent smokers did not differ with respect to age (57 ± 13 vs 64 ± 6 years) or pack-years (59 ± 25 vs 57 ± 21). There was no difference either in the microsomal (16.72 ± 3.33 mg g-1 tissue vs 15.67 ± 2.19 mg g tissue) and cytosolic (21.03 ± 2.20 mg g tissue vs 20.33 ± 1.70 mg g tissue) protein content. Microsomal and cytosolic EH activities were not significantly correlated each other either in the entire group of patients (r = 0.45) or in recent smokers and non-recent smokers separately (r 0.28 and r = 0.57, EH activity did not Microsomal respectively). differ between recent smokers (2.51 ± 0.93 nmol min 1 mg 1) and non-recent smokers (2.74 ± 1.10 nmol min-l mg-1). In contrast, cytosolic EH lower in recent smokers activity was (6.46 ± 1.79 pmol min mg-1) than in non-recent smokers (8.41 ± 2.09 pmol min mg-1, P < 0.05). This may suggest that recent smoking exposure selectively inhibits the activity of the cytosolic but not the microsomal EH. We searched for a possible relationship between exposure to tobacco smoke, expressed as the period since smoking ceased and the number of cigarettes smoked per day, with the activity of cytosolic EH. Cytosolic EH was correlated with the number of days that had elapsed since the smoking was stopped (r = 0.58, P < 0.05; Figure 1), as investigated up to 6
from Aldrich
(Milano, Italy).
Patients Nineteen patients submitted to open chest surgery for lung cancer or other non-neoplastic pulmonary diseases were included in the study. A standardized questionnaire was administered to each patient, in order to collect personal data with special regard to smoking habits. Patients were classified as ’recent smokers’ if they had smoked within the last month before surgery, and ’non-recent smokers’ were those who had stopped smoking more than 1 month before their
operations. Specimens of peripheral lung tissue were sampled at surgery. From lung cancer patients, tissue specimens were sampled from a normal portion of tissue as far away as possible from the tumor. Lung specimens were immediately frozen and were stored at -80°C until assayed. Subcellular isolation and assays
Aliquots (2.5 g) of lung tissue were minced and homogenized in five volumes of 0.25 M sucrose with a Polytron (Kinematica, Luzern, Switzerland). The homogenates were filtered on gauze, rehomogenized in a Potter-Elvehjem apparatus and then centrifuged at 9000 g for 15 min. The supernatant was then centrifuged at 105 000 g for 1 h. The supernatants were investigated as the cytosolic fractions. The resulting pellets were resuspended in 0.1 M Tris-HCl buffer (pH 7.4) containing 30% glycerol and studied as the microsomal fraction. Both microsomal and cytosolic fractions were stored at -80°C. Microsomal EH activity was determined according to the
Age,
Downloaded from het.sagepub.com at UNIV ARIZONA LIBRARY on June 1, 2015
=
significantly
101
Table 1 surgery.
Age,
sex,
smoking habit, diagnosis
and
epoxide hydrolase activity
in 19
patients
submitted to chest
Note. ’Recent smoker’ was defined as someone who was currently smoking or who had refrained from smoking for less than 30 d; ’non-recent smoker’ comprised those who had never smoked or who stopped it more than 30 days previously. EH cyt. cytosolic epoxide hydrolase; EH mic microsomal epoxide hydrolase =
=
Figure 1 Correlation between cytosolic epoxide hydrolase activity (ordinate) and the number of days of refraining from smoking (abscissa) in lung tissue from
Figure
smokers.
recent smokers.
months from surgery, with the lowest activity in patients who had been smoking on the day before surgery. Furthermore in recent smokers the inhibitory effect was dose-dependent, since the enzyme activity was inversely correlated with the number of cigarettes smoked per day (r = 0.85, P
some
