Counts of Slackia sp . strain NATTS in Intestinal Flora are Correlated to Serum Concentrations of Equol both in Prostate Cancer Cases and Controls in Japanese Men

Recently, researchers have focused on the protective effect of isoflavones, in particular, equol converted from daidzein, in the epidemiology of prostate cancer (Kurahashi et al., 2008; Park et al., 2009; Travis et al., 2012). Isoflavones are one of the phytoestrogens that are possible preventive factors in several hormone-dependent cancers such as prostate cancer and breast cancer (Zhu et al., 2011; Khan et al., 2012 ), cardiovascular disease (Wong et al., 2012; Usui et al., 2013), menopausal symptoms (Aso et al., 2012; Jenks et al., 2012), and osteoporosis(Scheiber et al., 2001; Tousen et al., 2011). Especially, genistein and daidzein, which are aglycones of isoflavones, were found to have potential roles in the prevention of prostate cancer

Generally, isoflavones have a binding affinity to estrogen β-receptors (Kuiper et al., 1998) and equol which is colonal bacterial metabolite of daidzein can bind to sex hormone-binding globulin.Equol displays the highest affinity to 5α-dihydrotestosterone, and inhibits the growth of prostate cancer cells in vitro and in vivo (Lund et al., 2011).Equol is believed to have a stronger protective effect than daidzein against development of prostate cancer (Lund et al., 2004).
In soybean foods, isoflavones exist in the aglycone (genistein, daidzein, and glycitein) and glucoside forms (Richelle et al., 2002;Setchell et al., 2002).When soybeans are taken in, glucoside forms are hydrolyzed into absorbable aglycones.Furthermore, daidzein is metabolized into equol by intestinal bacteria in the colon (Rowland et al., 2003).The ability to produce equol depends on the presence of certain intestinal bacteria (Rowland et al., 2003).The proportion of equol-producer varies from 30% to 60%, and is higher among Asians and the elderly (Akaza et al., 2004;Fujimoto et al., 2008).Equol-producing intestinal flora have been reported in vitro human fecal culture (Setchell et al., 2010;Sugiyama et al., 2013).A new equol-producing bacterium, Slackia sp.strain NATTS was recently isolated from a healthy human fecal culture by addition of prebiotic sorbose.Slackia sp.strain NATTS was found to transform daidzein into equol, and belong to genus Slackia family Coriobacteriaceae by 16S rRNA gene sequence-based analysis (Tsuji et al., 2010).Tsuji et al. (2012) showed that, in Slackia sp.strain NATTS, the daidzein-to-equol conversion reaction proceeded by the action of a series of three enzymes.Other several strains converting daidzein into equol have already been found by cultivation in vitro from human intestinal bacteria.For instance, Enterococcus faecium EPI1, Finegoldia magna EPI3 and Lactobacillus mucosae EPI2 (Decroos et al., 2005), Aldercreutzia equolifaciens (Maruo et al., 2008), Eggerthella sp.YY7918 (Yokoyama et al., 2008), Slackia sp.HE8 (Matthies et al., 2009) and Slackia equolifaciens strain DZE (Jin et al., 2010) were isolated and identified.In this case-control study, we examined the association of serum isoflavones, and Slackia sp.strain NATTS with prostate cancer risk among Japanese men.

Study population/design
This case-control study was performed from September 2011 to May 2013 at Sapporo Medical University Hospital.Informed consent was obtained from every study subject.This study was approved by ethics committee of Sapporo Medical University.
A total of 44 cases were recruited from inpatients at the Department of Urology.They were newly diagnosed with clinically localized prostate cancer, for first time no more than 1 year from the diagnosis, and all have undergone laparoscopic radical prostatectomy.
A total of 28 controls were selected from inpatients at the Department of Dermatology and Department of Cardiovascular, Renal and Metabolic Medicine of the same hospital.Disease categories were dermatosis, hypertension and cardiovascular disease in the controls.Exclusion criteria for controls had a clinical history of benign prostatic hypertrophy, other prostatic disease, malignant tumors, and a serum prostate specific antigen (PSA) concentration more than 4.0ng/ml.In addition, exclusion criteria for cases and controls were dietary restriction and having taken antibiotics within a month of the survey.
The assayed isoflavones were genistein, daidzein, glycitein and equol.Fasting blood samples were drawn before breakfast and all samples were subsequently analyzed at a single laboratory (SRL, Inc.Tokyo, Japan) under blinding in cases and controls.Serum concentrations of isoflavones in the samples were measured using triple quadrupole tandem liquid chromatographymass spectrometry (LC-MS/MS).Controls were measured with PSA concentrations.Fresh fecal samples collected from both cases and controls were analyzed at a single laboratory at the Yakult Central Institute for Microbiological Research, Tokyo, Japan.The 16S rRNA gene fragments of Slackia sp.strain NATTS and related strains were amplified from the total RNA extracted from the fecal samples in order to determine the number of species by RT-qPCR (reverse transcription-quantitative PCR) (Tsuji et al., 2010).Detection limits were less than 0.5ng/ml for all isoflavones concentrations and 3.9 log 10 cell/g for counts of Slackia sp.strain NATTS.The questionnaire included the following information such as age, body height, body weight, and smoking habits.

Statistical analysis
Odds ratios (ORs) and 95% confidence intervals (CIs) for the risk of prostate cancer were estimated by the median cut-off level of serum isoflavones concentration on the frequency distribution of controls using a logistic regression models.For serum equol concentration and counts of Slackia sp.strain NATTS, the lower categories comprised study subjects with amounts below the detection limit (<0.5 ng/ml and <3.9 log 10 cells/g, respectively).ORs and 95% CIs were adjusted with age, body mass index (BMI) and smoking status.Student's t-test, chi-squared test, Mann-Whitney U test, Fisher's exact test, and Spearman correlation coefficients were also applied.All analyses were performed using SPSS version 18.0 and a p<0.05 was considered statistically significant.

Results
Characteristics of prostate cancer cases and controls are shown in Table 1.Average age, average BMI, smoking status, and distributions of serum isoflavones concentration and counts of Slackia sp.strain NATTS were not significantly different between cases and controls.
Table 2  As shown in Table 3, Slackia sp.strain NATTS in feces of cases and controls were 34.1% and 25.0%, respectively.Among detectable subjects for counts of Slackia sp.strain NATTS, proportions of subjects for detectable concentration of equol in cases and controls were 80.0% and 85.7%, respectively, and were not significantly different between cases and controls.Among nondetectable subjects for counts of Slackia sp.strain NATTS, proportions of subjects for non-detectable concentration of equol in cases and controls were 86.2% and 66.7%, respectively, and were not significantly different between cases and controls.
Table 4 present comparisons of data for the Spearman's correlation coefficients in the associations between age, BMI, serum isoflavones and Slackia sp.strain NATTS, in cases and controls.Counts of Slackia sp.strain NATTS were significantly correlated with serum equol in cases (r s =0.639, p<0.01) and in controls (r s =0.572, p<0.01).Age was significantly negatively associated with BMI in cases (r s =-0.523, p<0.01), but not in controls.Serum concentrations of genistein and daidzein were significantly correlated with each other both in cases and controls (r s =0.828 and r s =0.796, p<0.01, respectively).Serum concentrations of genistein and glycitein were significantly correlated with each other both in cases and controls (r s =0.697 and r s =0.572, p<0.01, respectively).Serum concentrations of daidzein and glycitein were significantly correlated with each other both in cases and controls (r s =0.845 and r s =0.772, p<0.01, respectively).

Discussion
In this study, serum concentrations of genistein, daidzein, glycitein, and equol were not associated with risk of prostate cancer.Large prospective studies in Europe indicated that higher a plasma concentration of genistein was significantly associated with a lower risk of prostate cancer (Travis et al., 2009).However, plasma concentrations of daidzein and equol were not significantly associated with risk of prostate cancer (Travis et al., 2009).In a nested case-control study among Japanese men, high serum levels of isoflavones, especially equol, significantly tended to reduce the risk of prostate cancer (Ozasa et al., 2004).Also, in another nested case-control study, the highest levels of genistein and equol were significantly associated with a decreased risk of localized prostate cancer (Kurahashi et al., 2008).However, several previous epidemiological studies (Ward et al., 2008;Travis et al., 2012) showed that serum isoflavones concentrations were not associated with risk of prostate cancer.To  In this study, we found that counts of Slackia sp.strain NATTS in feces were significantly associated with serum equol concentrations both in cases and controls.Subjects with detectable counts of Slackia sp.strain NATTS had detectable equol in high proportions.These results have suggested that Slackia sp.strain NATTS have a potent equol-producing ability from daidzein.Tsuji et al. (2010) reported that the detection rate of Slackia sp.strain NATTS in the feces of 40 healthy volunteers were 33% in subjects under 40 years old and were 47% in subjects over 40 years old.In the present study, detection proportions of subjects for Slackia sp.strain NATTS in cases and controls were smaller than these percentages shown by Tsuji et al. (2010).
We found that correlation between serum genistein and daidzein were statistically significant, and were correlated with serum glycitein both in cases and controls.Ozasa et al. (2004) showed that serum genistein and daidzein were very strongly correlated with each other.Mori et al. (2008) reported that serum daidzein concentration in patients with prostate cancer were significantly correlated with serum concentration of genistein and glycitein.Our results were consistent with these findings in cases and controls.
Our study has also shown that serum concentration of genistein and daidzein are not related to serum equol concentration either in cases or controls.Mori et al. (2008) showed that the serum concentration of equol was not correlated with the serum concentration of daidzein, genistein or glycitein.Sawada et al. (2010) indicated that plasma genistein was not correlated with equol in Japanese men.These results were consistent with the results from our study, although Ozasa et al. (2004) showed that serum concentration of genistein and daidzein were moderately correlated with serum equol concentration in controls.
Several limitations must be considered in this study.First, the sample size of this case-control study was small, and it caused reduced statistical power to identify as a risk factor for prostate cancer.Second, blood samples were collected after the diagnosis of prostate cancer cases and controls, although any case with prostate cancer had not been treated with chemotherapy, radiation or hormonal therapy.Third, controls were recruited from inpatients, but not from the general population, and it may have induced a selection bias.
In conclusion, this study indicated that counts of Slackia sp.strain NATTS in feces were correlated to serum concentration of equol in Japanese men.Serum isoflavones concentrations were not associated with risk of prostate cancer.Further study is required to confirm this finding.

Table 1 . Characteristics of Prostate Cancer Cases and Controls a
shows clinical stage and pathologic Gleason score in prostate cancer cases.The largest proportion of clinical stage was T1a-c as 50.0%, and the largest Values are expressed as mean±SD or median [interquartile range]; b p for t-test, χ 2 test or Mann-Whitney U test; c Preoperative value; d Values below the detection limit (<0.5ng/ml) were regarded as zero; e Values below the detection limit (<3.9log 10 cell/g) were regarded as zero DOI:http://dx.doi.org/10.7314/APJCP.2014.15.6.2693Intestinal Slackia sp.strain NATTS Correlates with Serum Equol Concentrations in Japanese Men proportion of pathologic Gleason score was 3+4 as 43.2%. a

Table 3 . Proportion of Slackia sp. strain NATTS and Equol above and below the Detection Limits in Cases and Controls
*Fisher's exact test