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

https://doi.org/10.1016/j.envres.2019.109074Get rights and content

Highlights

  • CYP enzymes are involved in drug and environmental agent metabolism.

  • CYP1A2 and CYP2A6 activities are altered at menopause independently of aging.

  • We suggest altered metabolism of drugs and environmental agents at menopause.

Abstract

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 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 modulation of xenobiotics metabolized by this enzyme. This study provides evidence that the transition to menopause induces significant alterations in xenobiotic-metabolizing enzymes independent of chronological aging suggesting altered metabolism of pharmaceutical and environmental agents.

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 mid-40s 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 xenobiotic-metabolizing 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). 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; 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.

Section snippets

Subjects and study design

Data were obtained from 152 (women n = 84, men n = 68) non-smoking 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.,

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

Discussion

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

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].

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