Abstract
Several studies revealed that gut microbiota affects the hepatic drug-metabolizing enzyme cytochrome P450 (Cyp). We hypothesized that individual gut microbiota variations could contribute to CYP activity. Human flora-associated (HFA) mice are established from germ-free mice using human feces and are often used to determine the effect of the human gut microbiota on the host. This study generated two groups of HFA mice using feces from two healthy individuals. Then, the composition of gut microbiota and hepatic Cyp activity was compared to analyze the effects of gut microbiota in healthy individuals on hepatic Cyp activity. A principal coordinate analysis based on the UniFrac distance for the composition of the cecal and fecal microbiota revealed apparent differences between the recipient groups. Hepatic Cyp, which is a marked difference in Cyp3a activity and Cyp3a11 gene expression, was observed between the recipient groups. Cyp2c and Cyp1a activities did not differ between recipient groups, with significantly lower enzymatic activities in recipients than in germ-free mice. These results indicate that the human gut microbiota affects hepatic Cyp activity. Especially, human gut microbiota composition differences have a pronounced effect on Cyp3a activity via Cyp3a11 gene expression regulation. Therefore, human gut microbiota variations among individuals may affect numerous drug metabolism, leading to drug efficacy and toxicity.
INTRODUCTION
The human gut microbiota influences many aspects of host physiology such as metabolism, immune system, and brain function (Sommer and Bäckhed, 2013; O’Toole and Jeffery, 2015; Schroeder and Bäckhed, 2016). The relationship between gut microbiota and drug metabolism is progressively becoming clearer in recent years (Zimmermann et al., 2019; Collins and Patterson, 2020; Klünemann et al., 2021), and the gut microbiota could influence drug-metabolizing enzymes in the host liver as one mechanism (Collins and Patterson, 2020; Tsunoda et al., 2021). In particular, many reports revealed that the existence of gut microbiota affects the major drug-metabolizing enzyme hepatic Cytochrome P450 (Cyp) 3a in mice (Claus et al., 2011; Kuno et al., 2016; Jourová et al., 2017). CYP3A is the major isoform of CYP, and it plays an important role in the metabolism of approximately half of the marketed drugs (Zuber et al., 2002). Therefore, the presence of gut microbiota likely influences the metabolism of a plethora of drugs metabolized by CYP3A.
Considerable individual variation in gut microbiota (Eckburg et al., 2005; Faith et al., 2013) and CYPs, including CYP3A (Takagi et al., 2008; Naidoo et al., 2014), has been shown even among healthy individuals. We have reported that hepatic Cyp3a activity differs in mice normalized with feces from different breeder mice (Togao et al., 2021). Therefore, we hypothesized that individual gut microbiota variations among healthy individuals could contribute to CYP3A activity and result in individual differences in drug metabolism.
Human flora-associated (HFA) mice are established from germ-free mice using human feces, which help determine the accurate effect of the presence of human gut microbiota on the health of the host (Zhang et al., 2014; Lundberg et al., 2020). Moreover, HFA mice have been used to compare the effect of different compositions of human gut microbiota on the host (Hirayama and Itoh, 2005). However, to date, no studies have used HFA mouse models to examine the effects of individual differences in human gut microbiota on hepatic drug-metabolizing enzymes.
Thus, in this study, two groups of HFA mice were generated using feces from two healthy individuals to evaluate the effects of individual differences in human gut microbiota on hepatic Cyp activity.
MATERIALS AND METHODS
Chemicals
Tris-HCl at 1 M (pH 9.0), ethylenediaminetetraacetic acid (EDTA) at 0.5 M (pH 8.0), Tris-EDTA buffer, Tris-EDTA-saturated phenol, phenol/chloroform/isoamyl alcohol (25:24:1), sodium acetate at 3 M (pH 5.2), and 10% sodium dodecyl sulfate were obtained from Nippon Gene Co., Ltd. (Tokyo, Japan). Isopropanol, ethanol, sucrose, glycerol, Tris, EDTA-2K, potassium dihydrogen phosphate, and potassium hydrogen phosphate were obtained from Fujifilm Wako Pure Chemical Co. (Osaka, Japan).
Feces
Fecal materials from healthy individuals provided by Cantor BioConnect, Inc. (Santee, CA, USA) were purchased from BizCom Japan, Inc. (Tokyo, Japan). The fecal materials were obtained following an approved protocol after review and approval by the ethics committee at the Cantor BioConnect facility. The fecal materials were stored at −80°C immediately after collection and were transported in containers cooled by dry ice, and dry ice remained in the containers upon receipt. Once obtained, the fecal materials were stored at −80°C until use.
Animals
Male, 5-week-old germ-free BALB/cAJcl mice (n = 24) were obtained from CLEA Japan, Inc. (Tokyo, Japan) to generate two groups of HFA mice (Recipient A and Recipient B, n = 8/group) from two healthy donors (Donor A and Donor B, corresponding with Recipient A and Recipient B). Additionally, the other 8 mice remained germ-free and served as the control group (Control).
All mice were maintained in cages in corresponding group isolators and kept on a 12/12-hr light/dark cycle. Room temperature and humidity were maintained at 20°C–26°C and 30%–70%, respectively. Mice were provided ad libitum access to radiation-sterilized chow (FR-2 50 kGy, Funabashi Farm Co., Ltd., Chiba, Japan) and sterilized water.
All experiments using animals were performed under the supervision of the Institutional Animal Care and Use Committee of Yakult Central Institute and approved by the Director of the Yakult Central Institute (approval number: 21-097). All animals were cared for and used following a program accredited by the AAALAC International.
Treatments
The mice were acclimated to the environment for approximately 1 week. The recipient group isolators received a tube containing donor feces and a tube containing saline solution, then approximately 1 g of feces suspended in a 9-fold volume of saline solution and 0.2 mL of supernatant was orally administered to each mouse. The Control group was given 0.2 mL of saline solution. The mice were then reared for 4 weeks.
Feces were collected and mice were removed from each isolator at 10 weeks of age. Feces were immediately stored at −80°C until use. Then, the weighted animals were exsanguinated from the posterior vena cava and abdominal aorta under isoflurane anesthesia, and the liver and cecal contents were harvested. Livers and cecal contents were frozen in liquid nitrogen and then stored at −80°C until use.
Gut microbiota analysis
DNA was extracted from donor human feces and recipient mice’s cecal contents and feces using glass beads and phenol using a previously described method (Matsuki, 2006).
The V4 region of the bacterial 16S rRNA gene was amplified and sequenced using the primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) following a previously described method (Caporaso et al., 2012) with minor modifications (Togao et al., 2021). Shortly, bar-coded amplicons were generated using TB Green Premix Ex Taq II (Takara Bio, Shiga, Japan) with approximately 10 ng of template DNA. The thermal cycler program was as follows: 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec, 50°C for 30 sec, and 72°C for 40 sec and was immediately stopped before amplification reached a plateau. An AMPure XP Kit (Beckman Coulter, Krefeld, Germany) was used to purify the amplicons, and a QuantiFluor® dsDNA System (Promega, Madison, WI, USA) was used to quantify their concentrations. The amplicons were pooled in equimolar amounts and sequenced on a MiSeq system (Illumina, San Diego, CA, USA) with a MiSeq Reagent Kit v2 500 cycle (Illumina).
The feature table (Supplemental File 1) and the relative abundances at the phylum and genus levels of bacteria were obtained by processing the sequence data in QIIME2 (Bolyen et al., 2019) (version 2022. 4) with silva (version 138_1) as a reference database. The difference in composition based on the unweighted and weighted UniFrac distances was visualized by principal coordinate analysis. Differences in occupancy between recipients were determined via linear discriminant analysis (LDA) effect size (LEfSe) analysis (Segata et al., 2011) with the requirements of an LDA score of > 4.0 using the Galaxy application (http://huttenhower.sph.harvard.edu/galaxy/). Additionally, Faith’s phylogenetic diversity (Faith’s PD) and observed features were computed using 20,000 reads per sample as alpha diversity indices.
Cyp activities
An established method was used to prepare liver microsomes, and the total protein concentration was measured using a previously described method (Togao et al., 2020). Cyp3a, Cyp2b, Cyp2c, and Cyp1a activities in liver microsomes were measured using the P450-Glo™ Assay (Promega) following the manufacturer’s protocol and a previous report (Togao et al., 2020). In detail, liver microsomes and a luminogenic substrate (luciferin IPA for Cyp3a, luciferin 2B6 for Cyp2b, luciferin H for Cyp2c, and luciferin 1A2 for Cyp1a) in 0.2 M of potassium phosphate buffer (pH 7.4) were preincubated for 10 min at 37°C. Then, the mixtures were incubated for 10, 20, 30, or 10 min for Cyp3a, Cyp2b, Cyp2c, or Cyp1a activity, respectively, at 37°C by adding NADPH regeneration systems (Promega). Then, the luciferin detection reagent was added and stabilized for 20 min at room temperature. A LUMIstar OPTIMA (BMG LABTECH, Offenburg, Germany) was used to measure luminescence.
Gene expression levels in the liver
The extraction of mRNA from small liver pieces and generation of first-strand cDNA was performed using the previously mentioned methods (Togao et al., 2021). cDNA was amplified by real-time polymerase chain reaction using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Waltham, MA, USA) and the AB7500 system (Applied Biosystems). Cyp3a11, Cyp2b10, and Cyp2c29, which are the dominant mouse isoforms of Cyp3a, Cyp2b, and Cyp2c, respectively (Martignoni et al., 2006), were selected as target genes. Additionally, the pregnane X receptor (Pxr), which is a dominant Cyp3a transcriptional regulator (Yoshinari et al., 2006; di Masi et al., 2009; Qin and Wang, 2019); multidrug resistance-associated protein 3 (Mrp3) and organic anion transporting polypeptide 2 (Oatp2), which are target genes of Pxr (Ishii et al., 2014; Li et al., 2009); Constitutive androstane receptor (Car), which is a major Cyp2b and Cyp2c transcriptional regulator (di Masi et al., 2009; Fujino et al., 2016); glucocorticoid receptor (Gr) and hepatocyte nuclear factor 4 alpha (Hnf4α), which are Cyp3a transcriptional regulators (Yoshinari et al., 2006; Qin and Wang, 2019); and Aryl hydrocarbon receptor (Ahr), which is a major Cyp1a transcriptional regulator (Ayed-Boussema et al., 2012; Yamashita et al., 2021) were selected. The sequence of each primer is shown in Supplemental Table 1. The delta-delta Ct method was used to compute the relative mRNA levels. β-actin was used for internal control.
Statistical analysis
Permutational multivariate analysis of variance (PERMANOVA) computed using QIIME2 was used to evaluate the difference in composition based on the unweighted and weighted UniFrac distances between recipients. The Mann-Whitney U-test was used to compare alpha diversity indices between recipients. The Bonferroni-corrected t-test was used to compare hepatic Cyp activity and gene expression (delta Ct) among groups. A p-value of < 0.05 was considered statistically significant. Statistical analyses, except PERMANOVA, were computed using Bell Curve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan).
RESULTS
Gut microbiota
Bacterial composition in feces from human donors and cecal contents and feces from recipient mice are shown as cumulative bar charts (Fig. 1). Firmicutes at the phylum level was the most dominant phylum in both recipient groups, followed by Bacteroidota, and these two phyla share > 90% of the total. A decreased Firmicutes occupancy was found in both recipient groups compared to the donor groups. The genus Bacteroides at the genus level was the most dominant in both recipient groups, followed by Blautia, accounting for approximately half of the total.
The principal coordinate analysis based on the unweighted and weighted UniFrac distances was performed to visualize the variation of gut microbiota in feces from donors and cecal contents and feces from recipient mice (Fig. 2). Clear differences were found in bacterial microbiota composition between the recipients, and PERMANOVA analysis of both fecal and cecal contents revealed significant differences between the recipients.
The LEfSe analysis of the cecal contents of recipient mice is shown in Fig. 3. The genera Marvinbryantia and Fusicatenibacter of the family Lachnospiraceae showed significantly higher occupancy in Recipient A. The genera Bifidobacterium of the family Bifidobacteraceae, Clostridium innocuum group of the family Erysipelotrichaceae, Parabacteroides of the family Tannerellaceae, and Lachnoclostridium and Ruminococcus gnavus group of the family of Lachnospiraceae showed significantly higher occupancy in Recipient B compared with Recipient A. These tendencies observed in the cecum were similar to feces (data not shown).
Faith’s PD and observed features for alpha diversity of cecal contents of recipient mice are shown in Fig. 4. Both were significantly higher in Recipient A (median 8.3 and 132) than in Recipient B (median 5.5 and 67). These tendencies observed in the cecum were similar to feces (Supplemental Fig. 1). Additionally, both Faith’s PD and observed features were higher in Donor A (14.0 and 285) than in Donor B (8.1 and 131).
Hepatic Cyp activities
Hepatic Cyp activities are shown in Fig. 5. Cyp3a activity was significantly higher in both recipient mice than in the Control group. Furthermore, Cyp3a activity was significantly higher in the Recipient A group than in the Recipient B group.
No clear differences were found in Cyp2b activity among the groups for the other isoforms. Cyp2c and Cyp1a activities did not differ between recipients. Cyp2c and Cyp1a activities in both recipients were lower than in the Control group in contrast with Cyp3a activity.
Hepatic Cyp and related gene expressions
Cyp gene expression levels are shown in Fig. 6. The expression of Cyp3a11, which is a major isoform of mouse Cyp3a, was significantly higher in both recipient groups than in the Control group. Furthermore, the Cyp3a11 expression level was significantly higher in the Recipient A group than in the Recipient B group. The expression level of Cyp2b10, which is a major isoform of mouse Cyp2b, was significantly higher in the Recipient B group than in the Control group. No significant difference was found in the Cyp2c29 expression level, which is a major isoform of mouse Cyp2c, among any of the groups. The Cyp1a2 expression level, which is a major isoform of mouse Cyp1a, was significantly lower in both recipient groups than in the Control group.
Gene expression levels of nuclear receptors, which is the Cyp transcriptional regulators, are shown in Fig. 7. Pxr and Car were not significantly different among the groups; Gr was significantly lower in the Recipient B group than in the Control group. Hnf4α was significantly higher in the Recipient A group than in the Control group. Ahr was significantly lower in both recipient groups than in the Control group. Additionally, gene expression levels of Mrp3 and Oatp2, which are target genes of Pxr were not significantly different among the groups (Supplemental Fig. 2).
DISCUSSION
Previous studies revealed that gut microbiota could influence the hepatic Cyps, mainly Cyp3a (Claus et al., 2011; Selwyn et al., 2016; Jourová et al., 2017). However, no study revealed the effects of human gut microbiota composition differences on hepatic Cyps. Thus, this study used two groups of HFA mice generated by feces from two healthy individuals and revealed that human gut microbiota composition differences have a pronounced effect on hepatic Cyp3a activity.
Principal coordinate analysis based on unweighted and weighted UniFrac distances revealed apparent microbiota composition differences in both cecal contents and feces between the recipient groups. Moderate differences were found in the microbiota composition between donors and recipients, and this phenomenon was shown in a previous study (Zhang et al., 2014), which could be due to a species difference between humans and mice. LEfSe analysis confirmed the presence of several bacterial genera characteristics of each recipient group. For instance, some Lachnoclostridium species, which are characteristic in the Recipient B group, are known to produce secondary bile acid (Burgess et al., 2020) which might have Cyp3a-regulating potential (Toda et al., 2009a; Barretto et al., 2021). Additionally, clear differences were found in alpha diversity between recipients which have the same trends as donors. These results indicate that we established two groups of HFA mice models using feces from two healthy individuals with distinctly different cecal contents and fecal microbiota compositions, which inherited characteristics of donor gut microbiota.
Cyp3a activity was significantly higher in both recipient groups than in the Control group. This result is consistent with previous reports that the presence of gut microbiota enhances hepatic Cyp3a activity (Toda et al., 2009b; Selwyn et al., 2016; Togao et al., 2020). Furthermore, an apparent difference was found in Cyp3a activity between recipients. In this study, possible factors that may affect Cyp activity, such as environmental xenobiotics, food, and age (MacLeod et al., 2000), were strictly unified. Therefore, these results strongly suggest that human gut microbiota differences have a clear effect on Cyp3a activity. One of the comparable CYP3A differences is sex differences regarding the differences observed between the recipient mice in this study (approximately 2-3 fold). Sex differences are well known, and previous reports demonstrated 2-fold greater hepatic CYP3A4 activity in females than in males (Wolbold et al., 2003). Moreover, another study reported that females displayed an average of 20%–30% increased clearance for drugs that were CYP3A substrate (Greenblatt and von Moltke, 2008). Human CYP3A is an isoform involved in the metabolism of > 50% of the drugs on the market (Zuber et al., 2002). Hence, gut microbiota differences in humans may affect a lot of drug efficacy and toxicity through drug metabolisms by CYP3A.
Cyp2c activity was significantly lower in both recipient groups than in the Control group. In the past, studies comparing germ-free and SPF mice reported conflicting results regarding the effects of gut microbiota on Cyp2c (Claus et al., 2011; Selwyn et al., 2015). In our previous study, we reported that the Cyp2c activity was lower in the group of mice normalized with mice feces from certain breeders than that in control (germ-free) mice; however, this difference was not observed between groups of mice normalized with feces from other breeders and controls (Togao et al., 2021). Taken together, gut microbiota differences may have different effects on Cyp2c activity. The microbiota composition of the recipients in this study had a decreasing effect on Cyp2c activity because both recipient groups showed lower Cyp2c activity. Similarly, Cyp1a activity was significantly lower in both recipient groups than in the Control group. The effect of gut microbiota on Cyp1a has also been controversial (Toda et al., 2009b; Fu et al., 2017). Further studies, with a larger number of donors, are needed to elucidate the effects of gut microbiota on these isoforms.
Gene expression levels of Cyp3a11, which is the major isoform of Cyp3a, were significantly different between the recipients. The aforementioned study using mice conventionalized with feces of mice from different breeders (Togao et al., 2021) revealed no differences in Cyp3a11 gene expressions among recipients. Hence, the occupancy of the bacterial groups affecting the Cyp3a expression may have been markedly different between the recipients in this study, considering the differences in group trends in Cyp3a11 gene expression in the present study and the previous study. This study revealed that gut microbiota differences influence Cyp3a activity by regulating the Cyp3a11 gene expression level.
Among the nuclear receptors, Pxr is commonly believed to be the dominant transcriptional regulator of Cyp3a (di Masi et al., 2009; Qin and Wang, 2019). This study revealed no group differences for Pxr or Mrp3 and Oatp2 which are target genes of Pxr (Ishii et al., 2014; Li et al., 2009) and these were different from the group trends in Cyp3a activity and Cyp3a11 gene expression levels. These findings suggest that other transcriptional regulators could also be involved in enhancing Cyp3a activity in the present model. To date, metabolites derived from the gut microbiota that may have Cyp3a-inducing potential include lithocholic acid (Toda et al., 2009a), which is a secondary bile acid; indole propionic acid (Venkatesh et al., 2014), which is an indole metabolite. Since both of these metabolites were mediated by Pxr (Staudinger et al., 2001; Venkatesh et al., 2014), in this study, it is suggested that other metabolites were also involved in the induction of Cyp3a11.
As for other nuclear receptors with the potential to modulate Cyp3a (Yoshinari et al., 2006; Qin and Wang, 2019), this study revealed no group differences for Car while Gr was lower in the recipient B group than in the Control group. These results differed from the group trends in Cyp3a activity and Cyp3a11 gene expression levels. However, the expression of another nuclear receptor Hnf4α, which is a Cyp3a transcriptional regulator (Yoshinari et al., 2006; Qin and Wang, 2019), was higher in the Recipient A group than in the Control group. Additionally, the Recipient B group had higher levels compared to the Control group, and the overall trend was similar to that of Cyp3a activity and Cyp3a11 gene expression, although not significant. This study suggests that Hnf4α may contribute to the modification of Cyp3a activity by the human gut microbiota composition although few studies focused on the effect of the gut microbiota on Hnf4α (Jourová et al., 2020). Further studies are needed to evaluate the actual contribution of Hnf4α.
Additionally, Ahr was significantly lower in both recipient groups compared to the Control group, with a similar group trend to that of Cyp1a activity and Cyp1a2 gene expression levels. Generally, Ahr is a major Cyp1a transcriptional regulator (Ayed-Boussema et al., 2012; Yamashita et al., 2021). This suggests that the low Cyp1a activity and Cyp1a2 gene expression levels in the recipient group in this study were mediated by Ahr. Conflicting results on the effect of gut microbiota on Ahr were shown in previous studies (Toda et al., 2009b; Fu et al., 2017). However, the Ahr results and Cyp1a results showed similar group trends (Toda et al., 2009b; Fu et al., 2017). Taken together, the gut microbiota may have an inhibitory or inductive effect on Cyp1a via Ahr, depending on the composition, although the mechanism is unknown.
Generally, host species differences exist in the inducibility of drug-metabolizing enzymes dependent on nuclear receptor ligand responsiveness (Martignoni et al., 2006), and the response and extent of the effects of gut microbiota on CYPs may differ between humans and mice. Therefore, further studies are needed to evaluate the inducibility of human CYPs by gut microbiota.
In conclusion, the human gut microbiota influences Cyp activity. Especially, gut microbiota composition differences have a pronounced effect on Cyp3a activity. Therefore, gut microbiota composition differences in humans may affect the metabolic capacity of many drugs, leading to their efficacy and toxicity.
ACKNOWLEDGMENTS
The authors are particularly grateful for the technical assistance given by Tomonori Aida.
The authors would also like to thank Enago (www.enago.jp) for the English language review.
Conflict of interest
The authors declare that there is no conflict of interest.
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