Cytochrome P450IA1 activity in human myometrium and uterine leiomyoma: Influence of smoking

IOXICOIOGY [ IS}VIFR S('[ENTIFI( PUll[ D;}[I RN I R I [ A.N[)

Toxicology 83 (1993) 41-48

Cytochrome P450IA1 activity in human myometrium and uterine leiomyoma: Influence of smoking S. Lamminen *a, K. Pyykk6 b, M. Rorarius c, R. Tuimala a'd aDepartment of Clinical Sciences, University of Tampere Box 607, SF-33101 Tampere hDepartment of Pharmacology, University of Turku, SF-20520 Turku "Department of Anesthesiology. dDepartment of Gynecology and Obstetrics, Tampere University Hospital, SF-33521 Tampere, Finland (Received 25th September 1992: accepted 10th April 1993)

Abstract

A xenobiotic-metabolizing enzyme, the P450IA1 (aryl hydrocarbon hydroxylase; AHH) was measured fluorometrically in myometrium and in uterine leiomyoma tissues. The material included both myometrium and leiomyoma tissue samples from 62 leiomyoma patients and 22 tissue samples of normal myometrium obtained from leiomyoma-free patients undergoing hysterectomy. Among 62 leiomyoma patients there were 14 cigarette-smokers and 48 nonsmokers, and among 22 control patients 6 and 16, respectively. The P450IA1 activity of the leiomyomas was significantly (P < 0.0005) higher than that of the surrounding myometrium in both smoker and non-smoker groups. In smokers, myometrium an obvious induction of P450IA1 was seen. Smoking increased the P450IA1 activity also in the leiomyoma, but the increase was not statistically significant. Our study supports the idea that myometrial tissue can respond to xenobiotics from the environment.

Key words." P450IAI; Aryl hydrocarbon hydroxylase; Myometrium; Uterine leiomyoma; Smoking

1. Introduction Cytochrome P450 isoenzymes play a key role in the metabolism of many endogenous substrates as well as a wide variety of xenobiotics, such as drugs, * Corresponding author.

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S. Lamminen et al. /Toxicology 83 (1993) 41-48

chemical carcinogens and environmental pollutants (Pelkonen and Nebert, 1982). The products of CYP1A genes, P450IAI and P450IA2, are primarily responsible for the metabolic activation of a number of environmental chemicals (Nebert, 1979; Nebert et al., 1989). Of these isoenzymes, only P450IA1 seems to be inducible in human extrahepatic tissues (McLemor et al., 1990; Sesardiac et al., 1990). P450IA1 (aryl hydrocarbon hydroxylase; AHH) activity is induced by polycyclic aromatic hydrocarbons, including cigarette smoke, in various human cells and tissues (Busbee et al., 1972; Huberman and Sachs, 1973; Bast et al., 1974; McLemor et al., 1977; Pelkonen et al., 1979; Karki et al., 1981; Suolinna et al., 1982; Das et al., 1986; McLemor et al., 1990; Sesardic et al., 1990; Barnea and Avigdor, 1991). The general observation is that the induction of AHH shows great interindividual and tissue-dependent variation. The P450-isoenzyme (AHH) effectively metabolizes known procarcinogenic compounds of cigarette smoke. Numerous investigators have established a relationship between the inducibility of AHH and malignant tumors in the lung (McLemor et al., 1977; Kouri et al., 1982; Suolinna et al., 1982; Korsgaard et al., 1984). The etiology and the growth of uterine leiomyomas is still unclear, but generally it has been linked with ovarian steroids. On the other hand, recent epidemiologic results indicate that women who smoke have an early menopause and a reduced risk of uterine leiomyomas (Baron et al., 1990). In the present study we measured the P450IA1 in the uterine leiomyomas of smoking and non-smoking women compared to P450IA1 in their myometriums. Our aim was to ascertain an inducibility of P450IA1 in the benign tumors, leiomyomas, and to see whether a connection exists between P450IAI induction and the established decrease in the incidence of uterine leiomyomas among smoking women.

2. Materials and methods

Myometrium (n = 62) and leiomyoma (n = 62) samples were obtained from 62 women undergoing hysterectomy for symptomatic large myomas or bleeding disorders. Of these patients, 14 (23%) were cigarette-smokers and 48 (77%) non-smokers according to the interviews, made first by a nurse and followed by an anesthesiologist during the medical examination. As a control group 22 myometrium samples from leiomyoma-free hysterectomy patients were collected. The absence of small intramural leiomyomas was confirmed histologically. In the control group there were 6 (27%) smokers and 16 (73%) non-smokers. The median age of all patients was 47 years (S.D. 10.0). After removal, the tissue specimens were put into small plastic bags, immediately frozen in liquid nitrogen and stored at -70°C until used for the preparation of microsomes.

S. Lamminen et al. / Toxicology 83 (1993) 41-48

43

The use of human tissues in this study was approved by the Ethical Committee of the University Hospital of Tampere. 2.1. Preparation of microsomes Microsomes of both leiomyoma and myometrium tissues were prepared by a method described in detail elsewhere (Lamminen et al., 1993). Frozen tissue specimens were pulverised with a Braun dismembrator and suspended in cold 10 mM Tris-buffer (pH 7.4) containing 10% glycerol. The homogenates were centrifuged first at 105 000 x g at 4°C for 60 min. The pellet containing the cell organelles was resuspended in 0.1 M K-phosphate buffer (pH 7.4) and re-centrifuged at 13 000 x g at 4°C for 10 min. The supernatant containing the microsomal fraction was finally filtered through Sepharose CL-2B gel to improve the purity of microsomes (Lamminen et al., 1993; Pyykk6, 1983). The microsomal protein yield per gram of myometrium and leiomyoma tissue was 1.2 mg and 1.3 mg, respectively. The ultimate microsomal fraction was frozen and stored at -70°C until the assay of AHH activity. 2.2. Enzyme and protein assay The P450IA1 (AHH) activity was determined spectrofluorometrically by measuring NADPH-dependent benzo[a]pyrene (BP) hydroxylation by the method of Nebert and Gelboin (1968). The reaction mixture (1 ml) contained microsomal protein (0.2-1.0 mg) in 0.1 M K-phosphate buffer (pH 7.4), MgCI2, NADPH, and benzo[a]pyrene in methanol as substrate. The incubation was carried out at 37°C for 60 min in the dark. The enzyme activity is expressed as fmol product (equivalent to 3-OH-BP) formed per mg protein per min. The protein contents of the microsomal fractions were determined by the method modified by Peterson, (1977). The average protein contents of myometrial and leiomyomal microsomes were 0.41 mg/ml (S.D. 0.14) and 0.42 mg/ml (S.D. 0.17), respectively. 2.3. Statistics The statistic calculatios were carried out using the STATGRAPHICS software (Graphic Software Systems, STSC, Inc., Rockville, USA). Logarithmically transformed enzyme activities were normally distributed around the mean and the differences between the means of log P450IA 1 were tested by the two-tailed t-test; a P-value < 0.005 was considered statistically significant. 3. Results

The distribution of the P450IA1 enzyme activities was remarkably wide and highly skewed in both myometrium and leiomyoma samples (Fig. 1). In leiomyomas the enzyme activities were significantly higher than those

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in the surrounding myometrium, independent of the smoking habits (Table 1). The P450IA1 activity was significantly higher (P < 0.005) in myometrium among the cigarette smokers, compared to the non-smokers. The same trend was seen in leiomyomas, the difference not being statistically significant, however (Table 1).

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200

300

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Fig. 1. The frequency distribution of P450IA1 activity (A) in myometrium (n = 84) and (B) in leiomyoma (n = 62). Figure 1A includes also myometrium samples from control patients.

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S. Lamminen et al. / Toxicology 83 (1993) 41-48

Table 1 The P450IA1 activities in the paired myometrium and leiomyoma samples of smokers and non-smokers. Activities expressed as median values with their ranges. Differences are tested between the means of log P450IA1 of the groups. Tissue

n

P450IAI (fmol/min/mg prot) median

Myometrium Non-smokers Smokers Leiomyoma Non-smokers Smokers

log P450IAI

P-value

mean

S.D.

range

48 14

33.5 64.5

0-155 10-143

1.62" 1.85"

1.57 1.66

< 0.005

48 14

101.5 178.0

6-604 16-364

2.08* 2.27*

2.05 2.06

< 0.05

*P < 0.0005.

Differences in P450IA1 activities between the leiomyoma-free myometrium and the myometrium from leiomyoma patients were not observed (Table 2). 4. Discussion

Aryl hydrocarbon hydroxylase (AHH) which is associated primarily with the cytochrome P450IAI (Nebert, 1979; Newbert et al., 1989) effectively metabolizes known promutagenic and procarcinogenic compounds, also present in cigarette smoke, into ultimate carcinogenic forms (Pelkonen and Nebert, 1982). The existence and role of AHH in malignant tumors associated with smoking and other forms of cancer has been extensively studied (Kouri et al., 1982; Suolinna et al., 1982; Korsgaard et al., 1984; Karki et al., 1987; Pyykk6 et al., 1991). We focused our interest on P450IAI activity in the normal myometrium and a benign uterine neoplasm, leiomyoma. The induction of P450IA 1 is genetically controlled (Kellerman et al., 1973; Table 2 The median P450AI1 activity (mol/min/mg prot.) in the myometriums of leiomyoma patients and in the myometriums of the control patients

Myometrium of leiomyoma patient Leiomyoma-free myometrium

n

Non-smokers

n

Smokers

48 16

33.5 31.5

14 6

64.5 63.0

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Nebert and Gonzalez, 1987), and a wide interindividual variation in the enzyme activities - - also observed in our material - - is a frequent finding in investigations involving P450IA1 in human cells. As to the reproductive tissues, cigarette smoke has been demonstrated to induce P450IA1 in the placenta (Pelkonen et al., 1979; Suolinna et al., 1982; Sesardic et al., 1990; Barnea and Avigdor, 1991). In our study, an obvious induction of P450IA1 was seen in smokers' myometrium. Also, in leiomyomas the enzyme activity increased under the influence of cigarette smoke, but the induction effect seems to be quantitatively weaker than that in myometrium. Earlier, Senler et al. (1985) reported higher cyclohexane hydroxylase activity in uterine leiomyoma than in the myometrium. Cyclohexane is a cyclic hydrocarbon, which serves as a non-specific substrate for several forms of P450. However, the result of the study was concluded to be due to the different protein contents in microsomal suspensions of leiomyoma and myometrium. Our results demonstrate a marked induction of P450IAl-dependent AHH in cigarette smokers' myometrium and confirms the significantly higher AHH activity in leiomyomas as compared to the adjacent myometrium. These findings constitute evidence of a capability of uterine cells to metabolize xenobiotics and a capacity of the myometrium to respond to environmental pollutants. Women who smoke had considerably higher P450IAI activity in the myometrium as compared to non-smokers. According to epidemiologic findings, smoking decreases the risk of uterine leiomyomas (Baron et al., 1990). It remains unresolved whether the induction of P450IA 1 in myometrium has a preventive effect on neoplasmic growth. The leiomyoma of myometrial origin had remarkable high P450IA1 activity, although the enzyme activity in the leiomyoma patients' myometrium was equally as low as that in the leiomyoma-free myometrium. We assume that the high P45IA1 activity in the leiomyoma expresses a distinctive property of the stem cell of neoplasm because leiomyoma is considered to be a monoclonal proliferation from a specific muscle cell of myometrium (Townsend et al., 1970). Another interpretation is that the alternation of monooxygenase activity in leiomyoma occurs after the clonal proliferation. Smoking does not explain the P450IA1 level of leiomyomas or an assumed high activity of the stem cell, because the enzyme activities were high in leiomyoma independently of smoking habits. The relationship between this altered monooxygenase activity in the leiomyoma and its growth should be resolved by further investigations. 5. References

Barnea, E.R. and Avigdor, S. (1991). Aryl hydrocarbon hydroxylase activity in the firsttrimester human placenta: Induction by carcinogens and chemoprotectors. Gynecol. Obstet. Invest. 32, 4.

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