Altered activity of xenobiotic detoxifying enzymes at menopause – A cross-sectional study

Environmental Research 182 (2020) 109074

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Altered activity of xenobiotic detoxifying enzymes at menopause – A crosssectional study

T

Vassiliki Tsiokoua, Thomas Kilindrisb, Elias Begasa, Evangelos Kouvarasa, Demetrios Kouretasc, Eftihia K. Asprodinia,∗ a

Department of Pharmacology, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41500, Biopolis, Larissa, Greece Department of Medical Informatics, Faculty of Medicine, Department of Biochemistry-Biotechnology, School of Health Sciences, University of Thessaly, 41500, Biopolis, Larissa, Greece c Laboratory of Animal Physiology – Toxicology, Department of Biochemistry-Biotechnology, School of Health Sciences, University of Thessaly, 41500, Biopolis, Larissa, Greece b

A R T I C LE I N FO

A B S T R A C T

Keywords: Menopause CYP1A2 CYP2A6 XO NAT-2

Xenobiotic metabolism at menopause is an under-investigated topic, albeit women spend one-third of their life in the postmenopausal period. The present study examined the effect of menopause on the in vivo activities of CYP1A2, CYP2A6, xanthine oxidase (XO) and N-acetyltransferase-2 (NAT2) xenobiotic metabolizing enzymes. Enzyme activity was determined in 152 non-smoking volunteers following oral intake of a single dose of 200 mg caffeine and subsequent determination of caffeine metabolite ratios (CMRs) in a 6-h urine sample as follows: CYP1A2: (AFMU+1U+1X)/17U, CYP2A6: 17U/(17U + 17X), XO: 1U/(1U+1X) and NAT2: AFMU/(AFMU +1U+1X). CMRs among groups were analyzed using one-way ANOVA. Significantly lower CYP1A2 and higher CYP2A6 CMRs were observed in postmenopausal compared to premenopausal women and age-matched men. These changes could be attributed to menopause rather than chronological aging since an age-related effect was not observed in premenopausal women or men of any age group. XO CMRs were higher in postmenopausal women and men > 50 compared to premenopausal women and men < 50, respectively, suggesting an agerelated increase in XO activity. No significant alterations were discerned in NAT2 CMRs, in either slow- or rapidacetylators, indicating that menopause exerts minimal modulation of xenobiotics metabolized by this enzyme. This study provides evidence that the transition to menopause induces significant alterations in xenobioticmetabolizing enzymes independent of chronological aging suggesting altered metabolism of pharmaceutical and environmental agents.

1. Introduction Natural menopause, resulting from the loss of ovarian function and the progressive decline in circulating estrogen levels, does not occur at a discrete point in time, but is rather a process that begins around mid40s and, despite the considerable inter-individual variation, the average age of onset is estimated at 51 years (McKinlay et al., 2008). The transition to menopause instigates multiple metabolic changes independent of chronological aging. Indeed, menopause has been associated with important disease risks including alterations in lipid metabolism favoring an atherogenic lipoprotein profile (Auro et al., 2014; Wang et al., 2018), altered glucose metabolism (Slopien et al., 2018), increased cardiovascular disease (Appelman et al., 2015; Colpani et al.,

2018) and osteoporosis (Karlamangla et al., 2018). Despite the wealth of information on the metabolic changes emanating from the decline of estrogens at menopause, there is a striking paucity of data concerning the possible consequences of menopause on xenobiotic metabolism, although numerous studies have shown that the activity of xenobioticmetabolizing enzymes may be modulated by female reproductive hormones. Thus, CYP1A2 activity is inhibited by estrogen-containing oral contraceptives (OCs) (Rasmussen and Brøsen, 1996; Granfors et al., 2005), by hormonal replacement therapy (HRT) in pre- and postmenopausal women (Pollock et al., 1999; O'Connell et al., 2006), during pregnancy (Vistisen et al., 1992; Tsutsumi et al., 2001; Tracy et al., 2005) and at the late follicular phase (LFP) of the menstrual cycle (Nagata et al., 1997; Kamimori et al., 1999; Asprodini et al., 2019).

∗

Corresponding author. Department of Pharmacology, Faculty of Medicine, University of Thessaly, 41500, Biopolis, Larissa, Greece. E-mail addresses: [email protected] (V. Tsiokou), [email protected] (T. Kilindris), [email protected] (E. Begas), [email protected] (E. Kouvaras), [email protected] (D. Kouretas), [email protected] (E.K. Asprodini). https://doi.org/10.1016/j.envres.2019.109074 Received 22 November 2019; Received in revised form 20 December 2019; Accepted 20 December 2019 Available online 28 December 2019 0013-9351/ © 2020 Elsevier Inc. All rights reserved.

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Nehlig, 2018). Τo our knowledge, no study has thus far addressed the issue of the influence of menopause on the above-mentioned xenobiotic-metabolizing enzyme activities with the exception of previous reports presenting incidental evidence on the effect of menopause on CYP1A2 (Hong et al., 2004) and CYP2A6 (Benowitz et al., 2006) enzyme activity. Therefore, the present study was designed to examine the hypothesis that the activity of xenobiotic metabolizing enzymes is modulated at menopause, independent of chronological age. To this end, we used caffeine as a metabolic probe to assess CYP1A2, CYP2A6, XO and NAT2 enzyme CMRs in a cohort of postmenopausal healthy women and to compare these CMRs to premenopausal women and to age-matched men.

CYP2A6 activity is enhanced at LFP (Asprodini et al., 2019), during pregnancy (Dempsey et al., 2002), or following the use of OCs (Benowitz et al., 2006). Conversely, the activity of XO and NAT2 enzymes remain unaltered during pregnancy (Tsutsumi et al., 2001), following estrogen therapy (O'Connell et al., 2006), or after the use of OCs (Rasmussen and Brøsen, 1996). The cytochrome P450 (CYP) superfamily is one of the major metabolizing enzyme systems in humans. Within the CYP superfamily, isoenzymes belonging to enzyme families 1–3 are responsible for 70–80% of all phase I dependent oxidative metabolism of clinically used drugs and they participate in the metabolism of a wide range of structurally diverse substrates such as endogenous compounds and xenobiotic chemicals (Evans and Relling, 1999). CYP1A2 is almost exclusively expressed in the liver accounting for approximately 13% of its total content (Shimada et al., 1994; Faber et al., 2005). It has the highest catalytic activity for the 2- and 4-hydroxylations of estradiol and estrone (Yamazaki et al., 1998), and it is involved in the biotransformation of several pharmaceutical drugs, such as theophylline, clozapine and olanzapine (Faber et al., 2005; Pelkonen et al., 2008). CYP2A6 is a highly polymorphic enzyme, it is expressed in the liver. It is responsible for the biotransformation of nicotine and its metabolite cotinine, several drugs such as valproic acid and pilocarpine and compounds of toxicological significance such as nitrosamines and aflatoxin B1 (Pelkonen et al., 2008; Di et al., 2009; Tanner and Tyndale, 2017). Although cytochrome P450 mediates primarily detoxification reactions, certain substrates are metabolically activated following P450 metabolism, resulting in the generation of reaction products with increased toxicity or mutagenicity, thus, leading to increased cancer risk. For example, CYP1A2 interacts with many environmental chemicals (Pasanen et al., 1995; Lagueux et al., 1999; Kim et al., 2004; Mizukawa et al., 2015; Docea et al., 2017; Vaughan et al., 2019). Correspondingly, CYP2A6 is responsible for the mutagenic activation of essentially all tobacco-related N-nitrosamines examined (Kamataki et al., 2002); as a consequence, allelic variation leading to reduction of CYP2A6 activity has been associated with reduced risk for lung cancer (Kamataki et al., 2005). It is of note that members of the CYP1 subfamily have been suggested to be responsible for the metabolic activation of several flavonoids leading to the suppression of cancer cell proliferation (Tsatsakis et al., 2011; Wilsher et al., 2017; Surichan et al., 2018a, 2018b; Surichan et al., 2018a, 2018b). Xanthine oxidase (XO) is the rate-limiting enzyme in purine catabolism and can oxidize a variety of endogenous substrates (aldehydes, purines, pyrimidines and pteridines). It contributes to liver detoxification through the catabolism of aminopurines (such as 2-aminopurine), heterocyclic compounds (such as 4-hydroxypyrimidine and retinol) (Battelli et al., 2014) and xenobiotics (such as antiviral and anticancer agents) (Pritsos, 2000; Battelli et al., 2014). N-acetyltransferase-2 (NAT2) is a polymorphic enzyme involved in the acetylation of several drugs, such as procainamide, nitrazepam, clonazepam, and isoniazid (Evans, 1989), and in the metabolism of environmental carcinogens including aromatic and heterocyclic amines (Dupret and Rodrigues-Lima, 2005). Caffeine has been widely accepted as a metabolic probe for the simultaneous assessment of CYP1A2, CYP2A6, XO and NAT2 phenotypes by the use of caffeine metabolite ratios (Relling et al., 1992; Cascorbi et al., 1995; Asprodini et al., 1998; Begas et al., 2007; Hakooz, 2009; Nehlig, 2018). In humans, most of caffeine is 3N-demethylated by CYP1A2 to paraxanthine (1,7-dimethylxanthine, 17X) (Butler et al., 1989) which is bio-transformed to 1,7-dimethyluric acid (17U) by CYP2A6 and to 1-methylxanthine (1X) by CYP1A2. 1X is eventually converted to 1-methyluric acid (1U) by XO, whereas, a small portion of paraxanthine is metabolized to 5-acetylamino- 6-formylamino-3-methyluracil (AFMU) by NAT2 (Gu et al., 1992; Kot and Daniel, 2008) (Fig. 1). The safety, availability and ease of administration of caffeine have made it possible to conduct large population studies in which CYP1A2, CYP2A6, XO and NAT2 enzyme activities can be assessed and compared between different subgroups of subjects (Hakooz, 2009;

2. Materials and methods 2.1. Subjects and study design Data were obtained from 152 (women n = 84, men n = 68) nonsmoking volunteers aged between 19 and 80 yrs. All volunteers were healthy according to medical history, physical examination and recent blood tests. Female volunteers were grouped according to menopausal status into pre- (n = 37) and postmenopausal (n = 47) groups. Menopause was specified, retrospectively, as the lack of spontaneous menstruation for at least 12 consecutive months prior to the recruitment to the study (McKinlay et al., 2008). The mean age at menopause was 50.39 ± 4.23 yrs. In order to compare enzyme activities between age-matched men and women, men were grouped into two age groups, men < 50 yrs and men > 50 yrs. Subjects using oral contraceptives (OCs) or hormonal replacement therapy (HRT) were excluded from the study, as were participants who consumed medications or supplements that are known to interact with CYP1A2 (known substrates, inducers or inhibitors) for seven days before and during the study. A form regarding demographic and lifestyle data (age, weight, height, age at menopause, chronic diseases, medication intake, alcohol consumption) as well as history of occupational and environmental exposure was completed by trained personnel for all participants. Volunteers were requested to refrain from alcohol (Kalow and Tang, 1991) and broiled meat (Kall et al., 1996) for at least 48 h before the caffeine test and to limit the consumption of cruciferous and apiaceous vegetables as these foods are known to exert a moderate influence on CYP1A2 activity (Kall et al., 1996; Lampe et al., 2000; Peterson et al., 2009). They were also requested to abstain from methylxanthine-containing food or beverages for a minimum of 24 h prior to the day of the caffeine test. On the day of the test, they were administered a capsule containing 200 mg caffeine and continued the abstinence from methylxanthines until urine sample collection. Spot urine samples were obtained 6 h after caffeine intake in containers preloaded with 200 mL of 6Ν HCl (Fig. 2). The study was approved by the Ethics Committee of Larissa University Hospital, Greece (1862015/24227) and was carried out in accordance with the Declaration of Helsinki. The participants were informed of every detail of the scope of the study and written informed consent was obtained from all subjects before inclusion to the study.

2.2. Enzyme phenotyping CMRs were used as phenotypic indices for the assessment of the in vivo activity of the enzymes examined. CYP1A2, CYP2A6, XO and NAT2 activities were estimated by the ratios (AFMU+1U+1X)/17U (Campbell et al., 1987; Begas et al., 2007), 17U/(17U + 17X) (Grant et al., 1983; Begas et al., 2017), 1U/(1X+1U) (Kalow and Tang, 1991) and AFMU/(AFMU+1U+1X) (Rostami-Hodjegan et al., 1996; Begas et al., 2007, 2019; Asprodini et al., 2019), respectively. 2

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Fig. 1. The 3-demethylation pathway of caffeine metabolism in human liver. Caffeine (1,3,7-trimethylxanthine, 137X) is demethylated to paraxanthine (1,7dimethylxanthine, 17X) by CYP1A2. Paraxanthine is oxidized to 1,7-dimethyluric acid (17U) by CYP2A6 and is demethylated to 1-methylxanthine (1X) which is subsequently oxidized to 1-methyluric acid (1U) by XO. Paraxanthine is also acetylated to 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by NAT2 after C8–N9 bond scission. Caffeine metabolites participating in the calculation of metabolic molar ratios are shown in blue. Enzymes and metabolic molar ratios used as indices of enzyme activities are shown in red (Arnaud, 2011; Nehlig, 2018). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

2.3. Chemicals

2.4. Sample analysis

Caffeine metabolites 17X, 17U, and (1U) were purchased from Sigma (Steinheim, Germany); 1-methylxanthine (1X) was purchased from TCI-Europe (Zwijndrecht, Belgium); 5-Acetylamino-6-formylamino-3-methyluracil (AFMU) was kindly provided by Wolfgang Pfleiderer (University of Konstanz, Konstanz, Germany). The Internal Standard paracetamol (4-acetamidophenol) was purchased from Sigma (Steinheim, Germany). Chloroform (analytical grade) and methanol (HPLC grade) were purchased from Chem Lab (Zedelgem, Belgium). Isopropanol (analytical grade) and acetonitrile (HPLC grade) were purchased from Honeywell-Riedel-de Haën (Seelze, Germany). Hydrochloric acid was purchased from Honeywell-Fluka (Seelze, Germany) and acetic acid was purchased from Merck (Darmstadt, Germany).

Urine samples were acidified to pH 3.5 to ensure AFMU stability (Wong et al., 2002); 1 mL aliquots were stored at −20 °C until analysis. Urinary caffeine metabolites were quantified by reversed-phase highperformance liquid chromatography as previously described (Begas et al., 2007). Caffeine metabolites were isolated from 200 μL urine samples by liquid-liquid extraction using 6 mL chloroform/isopropanol solution (85/15, v/v). Separation was achieved using a Kromasil 100 C18 column (5 μm, 250 × 4.6 mm i. d.; Macherey-Nagel, Germany) operated at 30 °C. The mobile phase (0.1% acetic acid/methanol/acenonitrile 92/4/5, v/v) was delivered at 0.7 mL/min for 5min and at 1.1 mL/min from 5 to 20min. Metabolites were detected at 280 nm. Calibration curves for caffeine metabolites in urine were linear at concentrations of 10–400μΜ with R2 > 0.99. Interday coefficients of variation for AFMU, 1U, 1X, 17U and 17X for the low Quality Control (QC) samples (30 μM) ranged between 4.45 and 10.01% with bias Fig. 2. Schematic diagram of the caffeine protocol followed in the study. Volunteers were asked to restrict their diet from broiled meat and alcohol for 2 days prior to the day of the caffeine test. 24 h prior to the day of the caffeine test, volunteers were asked to abstain from caffeinecontaining foods and beverages (filled circle designated with “24”). On the third day of the protocol volunteers ingested a capsule containing 200 mg caffeine at 8:00 a.m.; a urine sample was collected 6 h later under continued methylxanthine abstinence.

3

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between −3.86 and 4.52% (n = 15). Interday coefficients of variation for AFMU, 1U, 1X, 17U and 17X for the high QC samples (300 μM) ranged between 3.88% and 7.97% with bias between −3.03 and 2.88% (n = 15).

postmenopausal women, men < 50 and men > 50). Using linear regression and testing for non-zero coefficients of the model, we found that the factor age was non-zero (b1 = −0.028, p = 0.004) only in postmenopausal women (y = −0.028x+4.56, R2 = 0.174; Fig. 3Bb1, solid line). Conversely, in premenopausal women, men < 50 and men > 50 the coefficient of age was zero denoting that age does not account for the observed variance of CYP1A2 activity; consequently, these groups were modelled by a horizontal line at the level of their mean CYP1A2 CMR value (Fig. 3Bb1 dashed lines in upper and lower panels). Since the transition to menopause occurs at a different age for every woman, we recalculated the linear model in postmenopausal women using their menopausal (years in menopause) rather than their chronological age. The new model (b1 = −0.024, p = 0.016) increased unexplained variance of CYP1A2 (y = −0.024x+3.09, R2 = 0.125). This paradox may be explained in view of published evidence indicating that the hormonal decline through menopause transition stages is not monotonic and it is established approximately 4 years after the onset of menopause (Luetters et al., 2007; Greendale et al., 2019). Subsequently, the linear model was calculated again using a truncated sample consisting of subjects with menopausal age > 4 years. The new model (b1 = −0.035, p = 0.005) reduced unexplained variance of CYP1A2 CMRs (y = −0.035x+3.30, R2 = 0.200; Fig. 3Bb1, inset). Furthermore, because postmenopausal women exhibited higher BMI compared to premenopausal women (Table 2), and in view of the possible influence of BMI on CYP1A2 activity (Tantcheva-Poór et al., 1999), we examined whether CYP1A2 CMRs are affected by BMI. Using linear regression and testing for non-zero coefficients we found that the factor BMI was non-zero (b1 = −0.035, p = 0.048) only in postmenopausal women (y = −0.035x+3.78, R2 = 0.086; Fig. 3Bb2). Conversely, in premenopausal women, men < 50 and men > 50 the coefficient of BMI was zero denoting that BMI does not account for the observed variance of CYP1A2 activity and, therefore, these groups were modelled by a horizontal line at the level of their mean CYP1A2 CMR value (Fig. 3Bb2 dashed lines in upper and lower panels). Finally, we explored whether reduction of unexplained CYP1A2 CMR variance could be attained by considering both BMI and age as co-factors. Thus, we examined the possible co-linearity of BMI and age in all groups using Pearson correlation. The correlation between BMI and age was moderate in premenopausal women (r = 0.418, p = 0.009; linear regression: y = 0.211x+16.03, R2 = 0.175) and weak in men < 50 (r = 0.323 p = 0.027; linear regression: y = 0.173x+20.46, R2 = 0.130; Fig. 1C, solid lines in upper and lower panels), whereas in postmenopausal women and men > 50 no such correlation was found (Fig. 3C, dashed lines in upper and lower panels). A linear regression model including both BMI and age as cofactors, calculated for postmenopausal women, resulted in non-significant coefficient values suggesting that these factors combined do not account for the observed variance of CYP1A2 activity. Overall, the data suggest that the lower CYP1A2 CMR values observed in postmenopausal women, compared to premenopausal women and men, may be attributed to menopause (R2 = 0.200) rather than chronological aging (R2 = 0.174) or their increased BMI (R2 = 0.086).

2.5. Statistical analysis All data are presented as mean ± standard deviation (SD). Enzyme CMRs were analyzed using the General Linear Model. Multiple group comparisons were examined using one-way-ANOVA and were p-corrected according to Bonferroni correction. The statistical difference between two independent groups (samples stratified by either sex or age) was examined by the two-tailed Student's t-test. CMR values for all enzymes tested exhibited normal distribution. Co-linearity of BMI and age in all groups was examined using Pearson correlation. Univariate linear regression was used to model CYP CMRs. All statistical analyses were performed using SPSS version 26 software (IBM, Armonk, NY). P values < 0.05 were considered statistically significant. 3. Results All enrolled volunteers (n = 152) completed the study. No subject reported adverse effects following caffeine intake. The mean ( ± SD) age of women (n = 84) was 52.29 ± 14.54 yrs (median 54.4, range 19–80) and that of men (n = 68) 50.01 ± 17.04 yrs (median 48.0, range 22–79). There was no statistically significant difference in terms of age between premenopausal women (38.65 ± 8.26 yrs) and men < 50 (35.94 ± 7.95 yrs, p > 0.05) and between postmenopausal women (63.02 ± 7.81 yrs) and men > 50 (65.88 ± 8.18 yrs, p > 0.05). Postmenopausal women were characterized by statistically higher BMI (28.12 ± 4.31 kg/m2) compared to premenopausal women (24.10 ± 4.30 kg/m2, p < 0.001). Conversely, no statistically significant difference in BMI was found between men < 50 (26.61 ± 3.59 kg/m2) and men > 50 (26.50 ± 2.87 kg/m2, p > 0.05; Table 1). Postmenopausal women exhibited 20.9% significantly lower CYP1A2 CMRs (2.80 ± 0.52) compared to all other groups, namely, premenopausal women (3.54 ± 0.76), men < 50 (3.50 ± 0.71) and men > 50 (3.40 ± 0.68, one-way ANOVA p < 0.001; Table 2, Fig. 3Aa1). As previous studies have reported that sex or age may influence CYP1A2 activity, we examined CYP1A2 CMRs in subjects stratified by either sex or age. Women exhibited statistically significant lower CYP1A2 CMRs (3.13 ± 0.74, n = 84) compared to men (3.46 ± 0.69, n = 68, p = 0.006; Fig. 3Aa2). Statistically significant lower CYP1A2 activity was also observed in the group age > 50 (3.04 ± 0.66, n = 79) compared to the group age < 50 (3.52 ± 0.73, n = 73, p < 0.001; Fig. 3Aa3). As postmenopausal women participate in both the “all women” (Fig. 3Aa2), and the “age > 50” groups (Fig. 3Aa3), we examined whether it is menopause, a joint feature of both sex and age, the key parameter that accounts for the observed reduction in CYP1A2 activity. To this end, we adopted a linear model y = b1 x + b0 to fit CYP1A2 CMR values in all four groups (pre-, Table 1 Demographic characteristics of the participants.

BMI (kg/m2)

Age (yrs)

Women premenopausal postmenopausal Men men < 50 men > 50

n

mean ± SD

median (range)

mean ± SD

median (range)

37 47

38.65 ± 8.26 63.02 ± 7.81

39 (19–50) 62 (49–80)

24.10 ± 4.30 28.12 ± 4.31*

23.1 (18.3–38.3) 26.8 (22.2–40.0)

36 32

35.94 ± 7.95 65.88 ± 8.18

37 (22–51) 68 (51–79)

26.61 ± 3.59 26.50 ± 2.87

25.4 (19.3–35.7) 26.6 (20.5–33.0)

Difference between postmenopausal and premenopausal women *p < 0.001. 4

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Table 2 CMR values reflecting enzyme activities in all groups studied. CYP1A2

CYP2A6

XO

NAT-2

n Women premenopausal postmenopausal Men men < 50 men > 50

n

slow-

n

fast-

37 47

3.54 ± 0.76 2.80 ± 0.52*

0.59 ± 0.13 0.67 ± 0.12**

0.53 ± 0.05 0.58 ± 0.08&

21 23

0.10 ± 0.03 0.11 ± 0.03

16 24

0.40 ± 0.06 0.40 ± 0.08

36 32

3.50 ± 0.71 3.40 ± 0.68

0.63 ± 0.12 0.62 ± 0.16

0.54 ± 0.07 0.58 ± 0.06

21 19

0.12 ± 0.03 0.12 ± 0.04

15 13

0.39 ± 0.10 0.38 ± 0.09

Difference between postmenopausal and premenopausal, men < 50 and men > 50, *p < 0.001. Difference between postmenopausal and premenopausal, **p < 0.022. Difference between postmenopausal and premenopausal, &p < 0.001 and men < 50, &p < 0.002.

specific conditions such as menstruation, pregnancy, or the use of OCs and HRT (Harris et al., 1995; Schwartz, 2007; Marazziti et al., 2013) has been adequately studied. The present study is the first to demonstrate lower CYP1A2 and higher CYP2A6 CMRs in postmenopausal compared to premenopausal women and age-matched men. These changes could be attributed to menopause rather than chronological aging since an age-related effect was not observed in premenopausal women or men of any age group. XO CMRs were higher in postmenopausal women and men > 50 compared to premenopausal women and men < 50, respectively, suggesting an age-related increase in XO activity. No significant alterations were discerned in NAT2 CMRs, in either slow- or rapid-acetylators, indicating that menopause exerts minimal xenobiotic metabolism catalysed by this enzyme. One of the problems in addressing the influence of menopause on xenobiotic metabolism lies in the difficulty of separating an effect of menopause from a background variation in enzyme activity owed to age and sex. Several other endogenous (genetic polymorphisms and hormone levels) and exogenous factors, such as exposure to environmental (Nims et al., 1992; Landi et al., 1999) and pharmaceutical (Mostafa et al., 1990) agents, diet (Lampe et al., 2000; Larsen and Brøsen, 2005), alcohol consumption (Kalow and Tang, 1991) and cigarette smoking (Gunes et al., 2009) have been also suggested to affect enzyme activity. The potential role of sex-related dimorphism in CYP1A2 activity, with women exhibiting lower values, is exemplified by numerous studies conducted in healthy subjects of different ethnicities and nationalities (Relling et al., 1992; Vistisen et al., 1992; Nakajima et al., 1994; Carrillo and Benitez, 1996; Rasmussen and Brøsen, 1996; Kashuba et al., 1998; Tantcheva-Poór et al., 1999; OuYang et al., 2000; Rasmussen et al., 2002; Bebia et al., 2004; Gunes et al., 2009), although no difference between sexes (Catteau et al., 1995; Chung et al., 2000; Begas et al., 2007) or even higher activity in women (Nafziger and Bertino, 1989) has also been reported. Furthermore, human liver microsomal CYP1A2 activity is lower in females than males (Parkinson et al., 2004), a finding supporting the clinical observation that female patients treated with the CYP1A2 substrates clozapine (Lane et al., 1999) and olanzapine (Kelly et al., 2006; Bigos et al., 2008) exhibit slower metabolic disposition compared to men. Similar to sex, the evidence of the influence of age on CYP1A2 activity is also conflicting with many studies suggesting decrease in CYP1A2 activity with age (Chung et al., 2000; Gunes et al., 2009), while others indicating no effect (Catteau et al., 1995; Simon et al., 2001; Bebia et al., 2004; Begas et al., 2007). With respect to CYP2A6 activity, women exhibit significantly higher nicotine metabolism (Zeman et al., 2002; Benowitz et al., 2006) and CYP2A6 hepatic expression (Yang et al., 2012) and higher caffeine (Nowell et al., 2002; Sinues et al., 2008) and coumarin (Rautio et al., 1992) metabolism compared to men, which though does not reach statistical significance. This differentiation points to sexual dimorphism in CYP2A6 metabolism. Conversely, other studies have reported that CYP2A6 activity is not influenced by sex in young subjects (Nakajima et al., 2006; Djordjevic et al., 2010).

In all panels horizontal dashed lines indicate mean CYP1A2 CMR values. CYP2A6 CMRs were significantly higher in postmenopausal (0.67 ± 0.12) compared to premenopausal (0.59 ± 0.13, one-way ANOVA p = 0.022) women; no statistically significant differences were detected among other groups (Table 2, Fig. 4A). Examination of CYP2A6 CMRs in subjects stratified by either sex or age demonstrated that there was no statistically significant difference between men (0.62 ± 0.14) and women (0.64 ± 0.13; p > 0.05) and that subjects in the group age > 50 (0.65 ± 0.14) had significantly higher values compared to the ones in group age < 50 (0.61 ± 0.12; p = 0.036) (Fig. 4B). In accordance to CYP1A2, since postmenopausal women participate in both the “all women” and the “age > 50” groups, we examined whether menopause accounts for the observed increase in CYP2A6 activity. A linear model, fitted to CYP2A6 CMR values in each of the four groups, revealed that the coefficient of factor age was nonzero (b1 = 0.003, p = 0.001) only in postmenopausal women (y = 0.003x+0.467, R2 = 0.127; Fig. 4C, solid line). Conversely, in premenopausal women, men < 50 and men > 50 the coefficient of age was zero, hence, these groups were modelled by a horizontal line at the level of their mean CYP1A2 CMR value (Fig. 4C, dashed lines in left and right panels). A linear model (b1 = 0.008, p = 0.004) recalculated in postmenopausal women, using a truncated sample consisting of subjects with menopausal age > 4 years, reduced unexplained variance of CYP2A6 (y = 0.008x+0.546, R2 = 0.205; data not shown). The values of XO CMRs were higher in postmenopausal (0.58 ± 0.08) compared to premenopausal women (0.53 ± 0.05, p < 0.001) and men < 50 (0.53 ± 0.06, p = 0.002). Men > 50 (0.58 ± 0.06) exhibited higher XO CMRs compared to men < 50 (p < 0.012) and premenopausal women (p = 0.003); no statistically significant difference was found between postmenopausal women and men > 50 and between premenopausal women and men < 50 (Fig. 5A). These data suggest that age modulates XO activity in both women and men. Indeed, examination of XO CMRs in subjects stratified by either sex or age demonstrated that there was no statistically significant difference between men (0.55 ± 0.06) and women (0.56 ± 0.07; p > 0.05) (Fig. 5B, left) and that subjects in the group age > 50 (0.58 ± 0.07) had significantly higher values compared to the ones in group age < 50 (0.53 ± 0.05; p < 0.001) (Fig. 5B, right; Table 2). Slow- and rapid-acetylator phenotypes were discriminated according to the cut-off value 0.25 which has been previously shown to separate slow-from rapid-acetylators within the Greek population (Begas et al., 2007). No statistically significant difference was detected among groups for both slow- and rapid acetylators (Fig. 6; Table 2).

4. Discussion The effect of menopause on xenobiotic metabolism is an important yet under-investigated topic, albeit the effect of several other female5

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Fig. 3. Box plots of CMRs reflecting CYP1A2 activity in healthy volunteers. A. a1. Menopausal women exhibit lower CYP1A2 activity compared to premenopausal women and age-matched men (p < 0.001, one-way ANOVA). a2. Subjects stratified by sex exhibit lower CYP1A2 activity in women compared to men (p < 0.006, Student's t-test); a3. Postmenopausal women grouped with men > 50 exhibit lower CYP1A2 activity compared to premenopausal women grouped with men < 50 (p < 0.001, Student's t-test). B. b1. Chronological age (y = −0.028x+4.56, R2 = 0.174), menopausal age (y = −0.035x+3.30, R2 = 0.200, inset) and BMI (y = −0.035x+3.78, R2 = 0.086; b2.) are predictors of CYP1A2 activity only in postmenopausal women, unlike in the rest of the groups studied. C. The correlation between BMI and age is moderate in premenopausal women (r = 0.418, p = 0.009; linear regression: y = 0.211x+16.03, R2 = 0.175) and weak in men < 50 (r = 0.323 p = 0.027; linear regression: y = 0.173x+20.46, R2 = 0.130, solid lines), whereas in postmenopausal women and men > 50 no such correlation is found (dashed lines). 6

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Fig. 4. Box plots of CMRs reflecting CYP2A6 activity in healthy volunteers. A. Comparison among groups reveal higher CYP2A6 CMRs in postmenopausal compared to premenopausal women (p < 0.022, one-way ANOVA). B. No difference between men and women is observed; conversely, the group age > 50 exhibit higher CYP2A6 CMRs compared to the group age < 50 (p = 0.036, Student's t-test). C. Age is a predictor of CYP2A6 activity only in postmenopausal women (y = 0.003x+0.467, R2 = 0.127), unlike in the rest of the groups studied. In left and right panels horizontal dashed lines indicate mean CYP1A2 CMR values.

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Fig. 5. Box plots of CMRs reflecting XO activity in healthy volunteers. A. Postmenopausal women and men > 50 exhibit significantly higher XO CMRs compared to premenopausal women and men < 50, respectively (one-way ANOVA). B. No difference between men and women is observed when subjects are stratified by sex (left panel); conversely, the group age > 50 exhibits higher XO CMRs compared to the group age < 50 (right panel, p < 0.001 Student's t-test).

women explained 17.4% and 20.0% of the observed variance, respectively. Similarly, chronological and menopausal age as predictors of CYP2A6 activity in postmenopausal women explained 12.7% and 20.5% of the observed variance, respectively. These results suggest that long-term decline of estrogens modulates the activity of the CYP enzymes examined in the present study. In view of the higher BMI in postvs premenopausal women in our sample and the negative effect of BMI on CYP1A2-dependent caffeine clearance reported in humans (Tantcheva-Poór et al., 1999), we examined the contribution of BMI in CYP1A2 variance. Our results showed that CYP1A2 activity is lower at increasing BMI values solely in postmenopausal women with BMI accounting for 8.6% of the observed variance (Fig. 3Bb2). The physiological significance of this observation is not clear and warrants further exploration. Interestingly, we observed that BMI increases monotonically with age in premenopausal women and men < 50 and levels out in postmenopausal women and men > 50 (Fig. 3C) suggesting that menopause transition does not result in BMI increases in accordance to previous reports (Crawford et al., 2000; Greendale et al., 2019). . Our finding of lower CYP1A2 and higher CYP2A6 activity at menopause, when a dramatic reduction of estrogens occurs, is intriguing, as there is a general consensus in the literature suggesting that these enzyme activity alterations occur under estrogen-rich rather than

The controversy observed around sex- and age-related variation in CYP enzyme activity among related studies may result from differences in the experimental design such as the size and the men/women ratio in the sample, the inducing effect of smoking or the age-range of the participants. Remarkably, although multiple studies have employed a wide age-range for both men and women (Vistisen et al., 1992; Catteau et al., 1995; Le Marchand et al., 1997; Tantcheva-Poór et al., 1999; Chung et al., 2000; Simon et al., 2001; Rasmussen et al., 2002; Bebia et al., 2004), none of these studies has considered menopause as a possible source of variation in enzyme activity. The hypothesis that menopause alters CYP1A2 activity is supported by a study conducted in a sizeable sample of pre- and postmenopausal women reporting significantly lower CYP1A2 phenotypic activity at menopause (Hong et al., 2004), whereas studies designed to address CYP2A6 activity at menopause are lacking. Our data suggest that menopause, a joint feature of the sex- and age-related groups, may explain both the sex- (Fig. 3Aa2) and the age-related (Fig. 3Aa3) reported disparities in CYP1A2 activity. In fact, if aging was the predominant factor accounting for lower CYP1A2 activity, a gradual decline in enzyme activity would have been expected not only in the postmenopausal group, but also in the rest of the groups studied (Fig. 3Bb1). Moreover, both chronological and menopausal age as predictors of CYP1A2 activity in postmenopausal 8

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Fig. 6. Box plots of CMRs reflecting NAT2 activity in healthy volunteers. No statistically significant difference is detected among groups in slow- (upper diagram) or fast- (lower diagram) acetylators.

expression of cytochrome P450 enzymes (Dhir et al., 2006) through interaction with nuclear hormone receptors (Kennedy, 2008), as has been shown for sex steroid hormones (Konstandi et al., 2013). Such an interaction is supported by evidence showing that GH administration induces CYP1A2 activity in healthy (Jürgens et al., 2002) or GH-deficient (Cheung et al., 1996) adults. Taken together, these reports and the present findings point out that although the mechanisms underlying alterations of CYP activities at menopause remain elusive, it is possible that these alterations are not a direct result of estradiol depletion, but rather a complex hormonal interplay in the endocrine system of postmenopausal women. The higher XO CMRs observed in both postmenopausal women and men > 50yrs, compared to premenopausal women and men < 50, respectively, suggest that XO activity is influenced by age rather than menopause. Indeed, an age-related increase in XO activity has been shown in human plasma and tissues from experimental animals (Aranda et al., 2007; Vida et al., 2011). However, other caffeine-based studies (Relling et al., 1992; Chung et al., 2000), or studies in tissue samples from patients undergoing hepatectomy or liver biopsy (Guerciolini

estrogens-poor circumstances. For example, the activity of CYP1A2 is inhibited by OCs (Granfors et al., 2005), estrogen replacement therapy (Pollock et al., 1999; O'Connell et al., 2006), pregnancy (Vistisen et al., 1992; Tsutsumi et al., 2001; Tracy et al., 2005) and at the late follicular phase (LFP) of the menstrual cycle (Asprodini et al., 2019). Correspondingly, CYP2A6 is up-regulated by estradiol (Higashi et al., 2007) and its activity is increased by OCs (Benowitz et al., 2006; Sinues et al., 2008), pregnancy (Dempsey et al., 2002) and at LFP of the menstrual cycle (Asprodini et al., 2019). Dramatic changes in other components of the endocrine system, beyond estradiol, that emerge at menopause may explain, at least in part, our findings. Growth Hormone (GH) is secreted by the pituitary gland in a sexually dimorphic manner (Winer et al., 1990) and is reduced with age in both sexes (Chertman et al., 2000). Women, however, are characterized by an abrupt loss of pulsatile GH release pattern and a greater age-related decline in both total and pulsatile GH secretion compared to the more modest decline in men (Ho et al., 1987; Chertman et al., 2000) suggesting that the transition to menopause is accompanied by profound alterations in GH secretion. It has been reported that GH is the predominant regulator of the 9

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et al., 1991) failed to detect an influence of age on XO activity. XO is known to play an essential role in several oxidative stress-related situations and aging has been associated with a progressive deregulation of homeostasis resulting from chronic oxidative stress (Liguori et al., 2018). Therefore, a plausible explanation for increased XO activity with aging could be the progressive senescence of the immune system (Deleidi et al., 2015), with concomitant increased production of proinflammatory cytokines, leading to upregulation of XO expression and activity (Battelli et al., 2014). The lack of sex-related difference in XO CMRs reported here agrees with previous caffeine-based population studies in healthy subjects (Rasmussen and Brøsen, 1996; Aklillu et al., 2003; Djordjevic et al., 2010), although other studies have reported higher XO phenotypic activity in healthy females (Relling et al., 1992; Djordjevic et al., 2012) or higher XO activity assessed in hepatic tissue obtained from male patients with normal liver function (Guerciolini et al., 1991). Finally, the absence of altered NAT2 phenotypic activity at menopause is consistent with previous studies using caffeine as a metabolic probe suggesting that aging- (Relling et al., 1992; Djordjevic et al., 2012) and sex-related differences (Relling et al., 1992; Vistisen et al., 1992; Rasmussen and Brøsen, 1996; Djordjevic et al., 2012) do not occur in NAT2 activity in humans. We were able to address methodological shortcomings evident in previous reports: first, the effect of smoking, conferring variable genotype-dependent inducibility on CYP1A2 activity (Gunes et al., 2009), was circumvented by recruiting solely non-smoking volunteers. Second, perimenopausal women were excluded from the study, thus, allowing a clear separation between pre- and postmenopausal status. On the other hand, the lack of verification of menopausal status through estradiol level determination may be considered as limitation of the study. Furthermore, due to the restriction of our sample to healthy subjects, the study may not be directly generalizable to postmenopausal patient populations as advanced age often co-exists with diseases and polypharmacy (Koren et al., 2019). Moreover, due to the small sample size, these findings should be interpreted with caution, and ideally reconfirmed in a larger study. The present study provides evidence that transition to menopause induces significant alterations in xenobiotic-metabolizing enzymes independent of chronological aging. Although the clinical relevance of the present data remains to be determined, our study provides a better understanding of pharmacokinetic alterations during menopause, forms a basis for assessment of risk upon exposure to foreign chemicals and indicates the need of optimizing therapeutic protocols followed in postmenopausal women. Future studies employing toxicological tests, focusing on the long-term, low-dose real-life exposure to mixtures of xenobiotics, may contribute to the elucidation of the balance between toxicity and detoxification resulting from altered CYP activity at menopause.

Asprodini, E., Tsiokou, V., Begas, E., Kilindris, T., Kouvaras, E., Samara, M., Messinis, I., 2019. Alterations in xenobiotic-metabolizing enzyme activities across menstrual cycle in healthy volunteers. J. Pharmacol. Exp. Ther. 368, 262–271. Asprodini, E.K., Zifa, E., Papageorgiou, I., Benakis, A., 1998. Determination of N-acetylation phenotyping in a Greek population using caffeine as a metabolic probe. Eur. J. Drug Metab. Pharmacokinet. 23, 501–506. Auro, K., Joensuu, A., Fischer, K., Kettunen, J., Salo, P., Mattsson, H., Niironen, M., Kaprio, J., Eriksson, J.G., Lehtimäki, T., Raitakari, O., Jula, A., Tiitinen, A., Jauhiainen, M., Soininen, P., Kangas, A.J., Kähönen, M., Havulinna, A.S., AlaKorpela, M., Salomaa, V., Metspalu, A., Perola, M., 2014. A metabolic view on menopause and ageing. Nat. Commun. 5, 4708. Battelli, M.G., Bolognesi, A., Polito, L., 2014. Pathophysiology of circulating xanthine oxidoreductase: new emerging roles for a multi-tasking enzyme. Biochim. Biophys. Acta 1502–1517 1842. Bebia, Z., Buch, S.C., Wilson, J.W., Frye, R.F., Romkes, M., Cecchetti, A., Chaves-Gnecco, D., Branch, R.A., 2004. Bioequivalence revisited: influence of age and sex on CYP enzymes. Clin. Pharmacol. Ther. 76, 618–627. Begas, E., Bounitsi, M., Kilindris, T., Kouvaras, E., Makaritsis, K., Kouretas, D., Asprodini, E.K., 2019. Effects of short-term saffron (Crocus sativus L.) intake on the in vivo activities of xenobiotic metabolizing enzymes in healthy volunteers. Food Chem. Toxicol. 130, 32–43. Begas, E., Kouvaras, E., Tsakalof, A., Papakosta, S., Asprodini, E.K., 2007. In vivo evaluation of CYP1A2, CYP2A6, NAT-2 and xanthine oxidase activities in a Greek population sample by the RP-HPLC monitoring of caffeine metabolic ratios. Biomed. Chromatogr. 21, 190–200. Begas, E., Tsioutsiouliti, A., Kouvaras, E., Haroutounian, S.A., Kasiotis, K.M., Kouretas, D., Asprodini, E., 2017. Effects of peppermint tea consumption on the activities of CYP1A2, CYP2A6, Xanthine Oxidase, N-acetyltranferase-2 and UDP-glucuronosyltransferases-1A1/1A6 in healthy volunteers. Food Chem. Toxicol. 100, 80–89. Benowitz, N.L., Lessov-Schlaggar, C.N., Swan, G.E., Jacob, P., 2006. Female sex and oral contraceptive use accelerate nicotine metabolism. Clin. Pharmacol. Ther. 79, 480–488. Bigos, K.L., Pollock, B.G., Coley, K.C., Miller, D.D., Marder, S.R., Aravagiri, M., Kirshner, M.A., Schneider, L.S., Bies, R.R., 2008. Sex, race, and smoking impact olanzapine exposure. J. Clin. Pharmacol. 48, 157–165. Butler, M.A., Iwasaki, M., Guengerich, F.P., Kadlubar, F.F., 1989. Human cytochrome P450PA (P-450IA2), the phenacetin O-deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenic arylamines. Proc. Natl. Acad. Sci. U.S.A. 86, 7696–7700. Campbell, M.E., Spielberg, S.P., Kalow, W., 1987. A urinary metabolite ratio that reflects systemic caffeine clearance. Clin. Pharmacol. Ther. 42, 157–165. Carrillo, J.A., Benitez, J., 1996. CYP1A2 activity, gender and smoking, as variables influencing the toxicity of caffeine. Br. J. Clin. Pharmacol. 41, 605–608. Cascorbi, I., Drakoulis, N., Brockmöller, J., Maurer, A., Sperling, K., Roots, I., 1995. Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated Caucasian individuals: correlation with phenotypic activity. Am. J. Hum. Genet. 57, 581–592. Catteau, A., Poisson, N., Bonaïti-Pellié, C., Bechtel, Y.C., Bechtel, P.R., 1995. A population and family study of CYP1A2 using caffeine urinary metabolites. Eur. J. Clin. Pharmacol. 47, 423–430. Chertman, L.S., Merriam, G.R., Kargi, A.Y., 2000. In: Endotext, Feingold, K.R., Anawalt, B., Boyce, A., Chrousos, G., Dungan, K., Grossman, A., Hershman, J.M., Kaltsas, G., Koch, C., Kopp, P., Korbonits, M., McLachlan, R., Morley, J.E., New, M., Perreault, L., Purnell, J., Rebar, R., Singer, F., Trence, D.L., Vinik, A., Wilson, D.P. (Eds.), Growth Hormone in Aging. MDText.com, Inc., South Dartmouth (MA). Cheung, N.W., Liddle, C., Coverdale, S., Lou, J.C., Boyages, S.C., 1996. Growth hormone treatment increases cytochrome P450-mediated antipyrine clearance in man. J. Clin. Endocrinol. Metab. 81, 1999–2001. Chung, W.G., Kang, J.H., Park, C.S., Cho, M.H., Cha, Y.N., 2000. Effect of age and smoking on in vivo CYP1A2, flavin-containing monooxygenase, and xanthine oxidase activities in Koreans: determination by caffeine metabolism. Clin. Pharmacol. Ther. 67, 258–266. Colpani, V., Baena, C.P., Jaspers, L., van Dijk, G.M., Farajzadegan, Z., Dhana, K., Tielemans, M.J., Voortman, T., Freak-Poli, R., Veloso, G.G.V., Chowdhury, R., Kavousi, M., Muka, T., Franco, O.H., 2018. Lifestyle factors, cardiovascular disease and all-cause mortality in middle-aged and elderly women: a systematic review and meta-analysis. Eur. J. Epidemiol. 33, 831–845. Crawford, S.L., Casey, V.A., Avis, N.E., McKinlay, S.M., 2000. A longitudinal study of weight and the menopause transition: results from the Massachusetts Women's Health Study. Menopause 7, 96–104. Deleidi, M., Jäggle, M., Rubino, G., 2015. Immune aging, dysmetabolism, and inflammation in neurological diseases. Front. Neurosci. 9. Dempsey, D., Jacob, P., Benowitz, N.L., 2002. Accelerated metabolism of nicotine and cotinine in pregnant smokers. J. Pharmacol. Exp. Ther. 301, 594–598. Dhir, R.N., Dworakowski, W., Thangavel, C., Shapiro, B.H., 2006. Sexually dimorphic regulation of hepatic isoforms of human cytochrome p450 by growth hormone. J. Pharmacol. Exp. Ther. 316, 87–94. Di, Y.M., Chow, V.D.-W., Yang, L.-P., Zhou, S.-F., 2009. Structure, function, regulation and polymorphism of human cytochrome P450 2A6. Curr. Drug Metabol. 10, 754–780. Djordjevic, N., Carrillo, J.A., Gervasini, G., Jankovic, S., Aklillu, E., 2010. In vivo evaluation of CYP2A6 and xanthine oxidase enzyme activities in the Serbian population. Eur. J. Clin. Pharmacol. 66, 571–578. Djordjevic, N., Carrillo, J.A., Roh, H.-K., Karlsson, S., Ueda, N., Bertilsson, L., Aklillu, E., 2012. Comparison of N-acetyltransferase-2 enzyme genotype-phenotype and xanthine oxidase enzyme activity between Swedes and Koreans. J. Clin. Pharmacol. 52,

Acknowledgments We thank all volunteers who participated in the study. The study was financially supported by the Research Committee of the University of Thessaly [Grant 4822]. References Aklillu, E., Carrillo, J.A., Makonnen, E., Bertilsson, L., Ingelman-Sundberg, M., 2003. Xanthine oxidase activity is influenced by environmental factors in Ethiopians. Eur. J. Clin. Pharmacol. 59, 533–536. Appelman, Y., van Rijn, B.B., Ten Haaf, M.E., Boersma, E., Peters, S.A.E., 2015. Sex differences in cardiovascular risk factors and disease prevention. Atherosclerosis 241, 211–218. Aranda, R., Doménech, E., Rus, A.D., Real, J.T., Sastre, J., Viña, J., Pallardó, F.V., 2007. Age-related increase in xanthine oxidase activity in human plasma and rat tissues. Free Radic. Res. 41, 1195–1200. Arnaud, M.J., 2011. Pharmacokinetics and metabolism of natural methylxanthines in animal and man. Handb. Exp. Pharmacol. 33–91.

10

Environmental Research 182 (2020) 109074

V. Tsiokou, et al.

543–551. Lagueux, J., Pereg, D., Ayotte, P., Dewailly, E., Poirier, G.G., 1999. Cytochrome P450 CYP1A1 enzyme activity and DNA adducts in placenta of women environmentally exposed to organochlorines. Environ. Res. 80, 369–382. Lampe, J.W., King, I.B., Li, S., Grate, M.T., Barale, K.V., Chen, C., Feng, Z., Potter, J.D., 2000. Brassica vegetables increase and apiaceous vegetables decrease cytochrome P450 1A2 activity in humans: changes in caffeine metabolite ratios in response to controlled vegetable diets. Carcinogenesis 21, 1157–1162. Landi, M.T., Sinha, R., Lang, N.P., Kadlubar, F.F., 1999. Human cytochrome P4501A2. IARC Sci. Publ. 173–195. Lane, H.Y., Chang, Y.C., Chang, W.H., Lin, S.K., Tseng, Y.T., Jann, M.W., 1999. Effects of gender and age on plasma levels of clozapine and its metabolites: analyzed by critical statistics. J. Clin. Psychiatry 60, 36–40. Larsen, J.T., Brøsen, K., 2005. Consumption of charcoal-broiled meat as an experimental tool for discerning CYP1A2-mediated drug metabolism in vivo. Basic Clin. Pharmacol. Toxicol. 97, 141–148. Le Marchand, L., Franke, A.A., Custer, L., Wilkens, L.R., Cooney, R.V., 1997. Lifestyle and nutritional correlates of cytochrome CYP1A2 activity: inverse associations with plasma lutein and alpha-tocopherol. Pharmacogenetics 7, 11–19. Liguori, I., Russo, G., Curcio, F., Bulli, G., Aran, L., Della-Morte, D., Gargiulo, G., Testa, G., Cacciatore, F., Bonaduce, D., Abete, P., 2018. Oxidative stress, aging, and diseases. Clin. Interv. Aging 13, 757–772. Luetters, C., Huang, M.-H., Seeman, T., Buckwalter, G., Meyer, P.M., Avis, N.E., Sternfeld, B., Johnston, J.M., Greendale, G.A., 2007. Menopause transition stage and endogenous estradiol and follicle-stimulating hormone levels are not related to cognitive performance: cross-sectional results from the study of women's health across the nation (SWAN). J. Women's Health 16, 331–344. Marazziti, D., Baroni, S., Picchetti, M., Piccinni, A., Carlini, M., Vatteroni, E., Falaschi, V., Lombardi, A., Dell'Osso, L., 2013. Pharmacokinetics and pharmacodinamics of psychotropic drugs: effect of sex. CNS Spectr. 18, 118–127. McKinlay, S.M., Brambilla, D.J., Posner, J.G., 2008. The normal menopause transition. Maturitas 61, 4–16. Mizukawa, H., Nomiyama, K., Kunisue, T., Watanabe, M.X., Subramanian, A., Iwata, H., Ishizuka, M., Tanabe, S., 2015. Organohalogens and their hydroxylated metabolites in the blood of pigs from an open waste dumping site in south India: association with hepatic cytochrome P450. Environ. Res. 138, 255–263. Mostafa, M.H., Sheweita, S.A., Abdel-Moneam, N.M., 1990. Influence of some anti-inflammatory drugs on the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content. Environ. Res. 52, 77–82. Nafziger, A.N., Bertino, J.S., 1989. Sex-related differences in theophylline pharmacokinetics. Eur. J. Clin. Pharmacol. 37, 97–100. Nagata, K., Ishitobi, K., Yamamoto, Y., Ikeda, T., Hori, S., Matsumoto, Y., Sasaki, T., 1997. Increased theophylline metabolism in the menstrual phase of healthy women. J. Allergy Clin. Immunol. 100, 39–43. Nakajima, M., Fukami, T., Yamanaka, H., Higashi, E., Sakai, H., Yoshida, R., Kwon, J.-T., McLeod, H.L., Yokoi, T., 2006. Comprehensive evaluation of variability in nicotine metabolism and CYP2A6 polymorphic alleles in four ethnic populations. Clin. Pharmacol. Ther. 80, 282–297. Nakajima, M., Yokoi, T., Mizutani, M., Shin, S., Kadlubar, F.F., Kamataki, T., 1994. Phenotyping of CYP1A2 in Japanese population by analysis of caffeine urinary metabolites: absence of mutation prescribing the phenotype in the CYP1A2 gene. Cancer Epidemiol. Biomark. Prev. 3, 413–421. Nehlig, A., 2018. Interindividual differences in caffeine metabolism and factors driving caffeine consumption. Pharmacol. Rev. 70, 384–411. Nims, R.W., Beebe, L.E., Dragnev, K.H., Thomas, P.E., Fox, S.D., Issaq, H.J., Jones, C.R., Lubet, R.A., 1992. Induction of hepatic CYP1A in male F344/NCr rats by dietary exposure to Aroclor 1254: examination of immunochemical, RNA, catalytic, and pharmacokinetic endpoints. Environ. Res. 59, 447–466. Nowell, S., Sweeney, C., Hammons, G., Kadlubar, F.F., Lang, N.P., 2002. CYP2A6 activity determined by caffeine phenotyping: association with colorectal cancer risk. Cancer Epidemiol. Biomark. Prev. 11, 377–383. O'Connell, M.B., Frye, R.F., Matzke, G.R., St Peter, J.V., Willhite, L.A., Welch, M.R., Kowal, P., LaValleur, J., 2006. Effect of conjugated equine estrogens on oxidative metabolism in middle-aged and elderly postmenopausal women. J. Clin. Pharmacol. 46, 1299–1307. Ou-Yang, D.S., Huang, S.L., Wang, W., Xie, H.G., Xu, Z.H., Shu, Y., Zhou, H.H., 2000. Phenotypic polymorphism and gender-related differences of CYP1A2 activity in a Chinese population. Br. J. Clin. Pharmacol. 49, 145–151. Parkinson, A., Mudra, D.R., Johnson, C., Dwyer, A., Carroll, K.M., 2004. The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes. Toxicol. Appl. Pharmacol. 199, 193–209. Pasanen, M., Lang, S., Kojo, A., Kosma, V.M., 1995. Effects of simulated nuclear fuel particles on the histopathology and CYP enzymes in the rat lung and liver. Environ. Res. 70, 126–133. Pelkonen, O., Turpeinen, M., Hakkola, J., Honkakoski, P., Hukkanen, J., Raunio, H., 2008. Inhibition and induction of human cytochrome P450 enzymes: current status. Arch. Toxicol. 82, 667–715. Peterson, S., Schwarz, Y., Li, S.S., Li, L., King, I.B., Chen, C., Eaton, D.L., Potter, J.D., Lampe, J.W., 2009. CYP1A2, GSTM1, and GSTT1 polymorphisms and diet effects on CYP1A2 activity in a crossover feeding trial. Cancer Epidemiol. Biomark. Prev. 18, 3118–3125. Pollock, B.G., Wylie, M., Stack, J.A., Sorisio, D.A., Thompson, D.S., Kirshner, M.A., Folan, M.M., Condifer, K.A., 1999. Inhibition of caffeine metabolism by estrogen replacement therapy in postmenopausal women. J. Clin. Pharmacol. 39, 936–940. Pritsos, C.A., 2000. Cellular distribution, metabolism and regulation of the xanthine

1527–1534. Docea, A.O., Vassilopoulou, L., Fragou, D., Arsene, A.L., Fenga, C., Kovatsi, L., Petrakis, D., Rakitskii, V.N., Nosyrev, A.E., Izotov, B.N., Golokhvast, K.S., Zakharenko, A.M., Vakis, A., Tsitsimpikou, C., Drakoulis, N., 2017. CYP polymorphisms and pathological conditions related to chronic exposure to organochlorine pesticides. Toxicology Reports 4, 335–341. Dupret, J.-M., Rodrigues-Lima, F., 2005. Structure and regulation of the drug-metabolizing enzymes arylamine N-acetyltransferases. Curr. Med. Chem. 12, 311–318. Evans, D.A., 1989. N-acetyltransferase. Pharmacol. Ther. 42, 157–234. Evans, W.E., Relling, M.V., 1999. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286, 487–491. Faber, M.S., Jetter, A., Fuhr, U., 2005. Assessment of CYP1A2 activity in clinical practice: why, how, and when? Basic Clin. Pharmacol. Toxicol. 97, 125–134. Granfors, M.T., Backman, J.T., Laitila, J., Neuvonen, P.J., 2005. Oral contraceptives containing ethinyl estradiol and gestodene markedly increase plasma concentrations and effects of tizanidine by inhibiting cytochrome P450 1A2. Clin. Pharmacol. Ther. 78, 400–411. Grant, D.M., Tang, B.K., Kalow, W., 1983. Variability in caffeine metabolism. Clin. Pharmacol. Ther. 33, 591–602. Greendale, G.A., Sternfeld, B., Huang, M., Han, W., Karvonen-Gutierrez, C., Ruppert, K., Cauley, J.A., Finkelstein, J.S., Jiang, S.-F., Karlamangla, A.S., 2019. Changes in body composition and weight during the menopause transition. JCI Insight 4. Gu, L., Gonzalez, F.J., Kalow, W., Tang, B.K., 1992. Biotransformation of caffeine, paraxanthine, theobromine and theophylline by cDNA-expressed human CYP1A2 and CYP2E1. Pharmacogenetics 2, 73–77. Guerciolini, R., Szumlanski, C., Weinshilboum, R.M., 1991. Human liver xanthine oxidase: nature and extent of individual variation. Clin. Pharmacol. Ther. 50, 663–672. Gunes, A., Ozbey, G., Vural, E.H., Uluoglu, C., Scordo, M.G., Zengil, H., Dahl, M.-L., 2009. Influence of genetic polymorphisms, smoking, gender and age on CYP1A2 activity in a Turkish population. Pharmacogenomics 10, 769–778. Hakooz, N.M.K., 2009. Caffeine metabolic ratios for the in vivo evaluation of CYP1A2, Nacetyltransferase 2, xanthine oxidase and CYP2A6 enzymatic activities. Curr. Drug Metabol. 10, 329–338. Harris, R.Z., Benet, L.Z., Schwartz, J.B., 1995. Gender effects in pharmacokinetics and pharmacodynamics. Drugs 50, 222–239. Higashi, E., Fukami, T., Itoh, M., Kyo, S., Inoue, M., Yokoi, T., Nakajima, M., 2007. Human CYP2A6 is induced by estrogen via estrogen receptor. Drug Metab. Dispos. 35, 1935–1941. Ho, K.Y., Evans, W.S., Blizzard, R.M., Veldhuis, J.D., Merriam, G.R., Samojlik, E., Furlanetto, R., Rogol, A.D., Kaiser, D.L., Thorner, M.O., 1987. Effects of sex and age on the 24-hour profile of growth hormone secretion in man: importance of endogenous estradiol concentrations. J. Clin. Endocrinol. Metab. 64, 51–58. Hong, C.-C., Tang, B.-K., Rao, V., Agarwal, S., Martin, L., Tritchler, D., Yaffe, M., Boyd, N.F., 2004. Cytochrome P450 1A2 (CYP1A2) activity, mammographic density, and oxidative stress: a cross-sectional study. Breast Cancer Res. 6, R338–R351. Jürgens, G., Lange, K.H.W., Reuther, L.Ø., Rasmussen, B.B., Brøsen, K., Christensen, H.R., 2002. Effect of growth hormone on hepatic cytochrome P450 activity in healthy elderly men. Clin. Pharmacol. Ther. 71, 162–168. Kall, M.A., Vang, O., Clausen, J., 1996. Effects of dietary broccoli on human in vivo drug metabolizing enzymes: evaluation of caffeine, oestrone and chlorzoxazone metabolism. Carcinogenesis 17, 793–799. Kalow, W., Tang, B.K., 1991. Use of caffeine metabolite ratios to explore CYP1A2 and xanthine oxidase activities. Clin. Pharmacol. Ther. 50, 508–519. Kamataki, T., Fujieda, M., Kiyotani, K., Iwano, S., Kunitoh, H., 2005. Genetic polymorphism of CYP2A6 as one of the potential determinants of tobacco-related cancer risk. Biochem. Biophys. Res. Commun. 338, 306–310. Kamataki, T., Fujita, K., Nakayama, K., Yamazaki, Y., Miyamoto, M., Ariyoshi, N., 2002. Role of human cytochrome P450 (CYP) in the metabolic activation of nitrosamine derivatives: application of genetically engineered Salmonella expressing human CYP. Drug Metab. Rev. 34, 667–676. Kamimori, G.H., Joubert, A., Otterstetter, R., Santaromana, M., Eddington, N.D., 1999. The effect of the menstrual cycle on the pharmacokinetics of caffeine in normal, healthy eumenorrheic females. Eur. J. Clin. Pharmacol. 55, 445–449. Karlamangla, A.S., Burnett-Bowie, S.-A.M., Crandall, C.J., 2018. Bone health during the menopause transition and beyond. Obstet. Gynecol. Clin. N. Am. 45, 695–708. Kashuba, A.D.M., Bertino, J.S., Kearns, G.L., Leeder, J.S., James, A.W., Gotschall, R., Nafziger, A.N., 1998. Quantitation of three-month intraindividual variability and influence of sex and menstrual cycle phase on CYP1A2, N-acetyltransferase-2, and xanthine oxidase activity determined with caffeine phenotyping. Clin. Pharmacol. Therapeut. 63, 540–551. Kelly, D.L., Richardson, C.M., Yu, Y., Conley, R.R., 2006. Plasma concentrations of highdose olanzapine in a double-blind crossover study. Hum. Psychopharmacol. 21, 393–398. Kennedy, M.J., 2008. Hormonal regulation of hepatic drug metabolizing enzyme activity during adolescence. Clin. Pharmacol. Ther. 84, 662–673. Kim, Y.-D., Todoroki, H., Oyama, T., Isse, T., Matsumoto, A., Yamaguchi, T., Kim, H., Uchiyama, I., Kawamoto, T., 2004. Identification of cytochrome P450 isoforms involved in 1-hydroxylation of pyrene. Environ. Res. 94, 262–266. Konstandi, M., Cheng, J., Gonzalez, F.J., 2013. Sex steroid hormones regulate constitutive expression of Cyp2e1 in female mouse liver. Am. J. Physiol. Endocrinol. Metab. 304, E1118–E1128. Koren, G., Nordon, G., Radinsky, K., Shalev, V., 2019. Clinical pharmacology of old age. Expert Rev. Clin. Pharmacol. 12, 749–755. Kot, M., Daniel, W.A., 2008. The relative contribution of human cytochrome P450 isoforms to the four caffeine oxidation pathways: an in vitro comparative study with cDNA-expressed P450s including CYP2C isoforms. Biochem. Pharmacol. 76,

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Environmental Research 182 (2020) 109074

V. Tsiokou, et al.

Tracy, T.S., Venkataramanan, R., Glover, D.D., Caritis, S.N., National Institute for Child Health and Human Development Network of Maternal-Fetal-Medicine Units, 2005. Temporal changes in drug metabolism (CYP1A2, CYP2D6 and CYP3A Activity) during pregnancy. Am. J. Obstet. Gynecol. 192, 633–639. Tsatsakis, A.M., Androutsopoulos, V.P., Zafiropoulos, A., Babatsikou, F., Alegakis, T., Dialyna, I., Tzatzarakis, M., Koutis, C., 2011. Associations of xenobiotic-metabolizing enzyme genotypes PON1Q192R, PON1L55M and CYP1A1*2A MspI with pathological symptoms of a rural population in south Greece. Xenobiotica 41, 914–925. Tsutsumi, K., Kotegawa, T., Matsuki, S., Tanaka, Y., Ishii, Y., Kodama, Y., Kuranari, M., Miyakawa, I., Nakano, S., 2001. The effect of pregnancy on cytochrome P4501A2, xanthine oxidase, and N-acetyltransferase activities in humans. Clin. Pharmacol. Ther. 70, 121–125. Vaughan, A., Stevanovic, S., Jafari, M., Rahman, M., Bowman, R.V., Fong, K.M., Ristovski, Z., Yang, I.A., 2019. The effect of diesel emission exposure on primary human bronchial epithelial cells from a COPD cohort: N-acetylcysteine as a potential protective intervention. Environ. Res. 170, 194–202. Vida, C., Rodríguez-Terés, S., Heras, V., Corpas, I., De la Fuente, M., González, E., 2011. The aged-related increase in xanthine oxidase expression and activity in several tissues from mice is not shown in long-lived animals. Biogerontology 12, 551–564. Vistisen, K., Poulsen, H.E., Loft, S., 1992. Foreign compound metabolism capacity in man measured from metabolites of dietary caffeine. Carcinogenesis 13, 1561–1568. Wang, Q., Ferreira, D.L.S., Nelson, S.M., Sattar, N., Ala-Korpela, M., Lawlor, D.A., 2018. Metabolic characterization of menopause: cross-sectional and longitudinal evidence. BMC Med. 16, 17. Wilsher, N.E., Arroo, R.R., Matsoukas, M., Tsatsakis, A.M., Spandidos, D.A., Androutsopoulos, V.P., 2017. Cytochrome P450 CYP1 metabolism of hydroxylated flavones and flavonols: selective bioactivation of luteolin in breast cancer cells. Food Chem. Toxicol. 110, 383–394. Winer, L.M., Shaw, M.A., Baumann, G., 1990. Basal plasma growth hormone levels in man: new evidence for rhythmicity of growth hormone secretion. J. Clin. Endocrinol. Metab. 70, 1678–1686. Wong, P., Villeneuve, G., Tessier, V., Banerjee, K., Nedev, H., Jean-Claude, B.J., LeylandJones, B., 2002. Stability of 5-acetamido-6-formylamino-3-methyluracil in buffers and urine. J. Pharm. Biomed. Anal. 28, 693–700. Yamazaki, H., Shaw, P.M., Guengerich, F.P., Shimada, T., 1998. Roles of cytochromes P450 1A2 and 3A4 in the oxidation of estradiol and estrone in human liver microsomes. Chem. Res. Toxicol. 11, 659–665. Yang, L., Li, Y., Hong, H., Chang, C.-W., Guo, L.-W., Lyn-Cook, B., Shi, L., Ning, B., 2012. Sex differences in the expression of drug-metabolizing and transporter genes in human liver. J. Drug Metab. Toxicol. 3, 1000119. Zeman, M.V., Hiraki, L., Sellers, E.M., 2002. Gender differences in tobacco smoking: higher relative exposure to smoke than nicotine in women. J. Women's Health Gend. Based Med. 11, 147–153.

oxidoreductase enzyme system. Chem. Biol. Interact. 129, 195–208. Rasmussen, B.B., Brix, T.H., Kyvik, K.O., Brøsen, K., 2002. The interindividual differences in the 3-demthylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors. Pharmacogenetics 12, 473–478. Rasmussen, B.B., Brøsen, K., 1996. Determination of urinary metabolites of caffeine for the assessment of cytochrome P4501A2, xanthine oxidase, and N-acetyltransferase activity in humans. Ther. Drug Monit. 18, 254–262. Rautio, A., Kraul, H., Kojo, A., Salmela, E., Pelkonen, O., 1992. Interindividual variability of coumarin 7-hydroxylation in healthy volunteers. Pharmacogenetics 2, 227–233. Relling, M.V., Lin, J.S., Ayers, G.D., Evans, W.E., 1992. Racial and gender differences in N-acetyltransferase, xanthine oxidase, and CYP1A2 activities. Clin. Pharmacol. Ther. 52, 643–658. Rostami-Hodjegan, A., Nurminen, S., Jackson, P.R., Tucker, G.T., 1996. Caffeine urinary metabolite ratios as markers of enzyme activity: a theoretical assessment. Pharmacogenetics 6, 121–149. Schwartz, J.B., 2007. The current state of knowledge on age, sex, and their interactions on clinical pharmacology. Clin. Pharmacol. Ther. 82, 87–96. Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., Guengerich, F.P., 1994. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther. 270, 414–423. Simon, T., Becquemont, L., Hamon, B., Nouyrigat, E., Chodjania, Y., Poirier, J.M., FunckBrentano, C., Jaillon, P., 2001. Variability of cytochrome P450 1A2 activity over time in young and elderly healthy volunteers. Br. J. Clin. Pharmacol. 52, 601–604. Sinues, B., Fanlo, A., Mayayo, E., Carcas, C., Vicente, J., Arenaz, I., Cebollada, A., 2008. CYP2A6 activity in a healthy Spanish population: effect of age, sex, smoking, and oral contraceptives. Hum. Exp. Toxicol. 27, 367–372. Slopien, R., Wender-Ozegowska, E., Rogowicz-Frontczak, A., Meczekalski, B., ZozulinskaZiolkiewicz, D., Jaremek, J.D., Cano, A., Chedraui, P., Goulis, D.G., Lopes, P., Mishra, G., Mueck, A., Rees, M., Senturk, L.M., Simoncini, T., Stevenson, J.C., Stute, P., Tuomikoski, P., Paschou, S.A., Anagnostis, P., Lambrinoudaki, I., 2018. Menopause and diabetes: EMAS clinical guide. Maturitas 117, 6–10. Surichan, S., Arroo, R.R., Ruparelia, K., Tsatsakis, A.M., Androutsopoulos, V.P., 2018a. Nobiletin bioactivation in MDA-MB-468 breast cancer cells by cytochrome P450 CYP1 enzymes. Food Chem. Toxicol. 113, 228–235. Surichan, S., Arroo, R.R., Tsatsakis, A.M., Androutsopoulos, V.P., 2018b. Tangeretin inhibits the proliferation of human breast cancer cells via CYP1A1/CYP1B1 enzyme induction and CYP1A1/CYP1B1–mediated metabolism to the product 4′ hydroxy tangeretin. Toxicol. In Vitro 50, 274–284. Tanner, J.-A., Tyndale, R.F., 2017. Variation in CYP2A6 activity and personalized medicine. J. Personalized Med. 7. Tantcheva-Poór, I., Zaigler, M., Rietbrock, S., Fuhr, U., 1999. Estimation of cytochrome P450 CYP1A2 activity in 863 healthy Caucasians using a saliva-based caffeine test. Pharmacogenetics 9, 131–144.

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