chemicals : bisphenol a and high - performance liquid chromatography ( hplc ) 
 grade methanol were purchased from kanto chemical co. ( tokyo , japan ) . the glucuronide 
 metabolites of bisphenol analogs were purified from bile after rat liver perfusion with 7.5 
 mole bisphenol a , bisphenol f and bisphenol af   and were quantified by hplc by determining the difference between 
 untreated and beta - glucuronidase - treated samples , which were used as standards . animals : ten - week old c57bl/6 mice ( pregnant and neonatal mice with 
 mothers ) were purchased from sankyo lab co. ( tokyo , japan ) . animals were housed individually 
 under a 12/12-hr light / dark cycle and had ad libitum access to water and 
 food . all of the animals were treated according to the laboratory animal control guidelines 
 at rakuno gakuen university , which are based on the directive 2010/63/eu revising directive 
 86/609/eec on the protection of animals used for scientific purposes ( ethics committee 
 protocol approval number es23a07 , approved jan 13 , 2012 ) . tissue collections : dams and neonatal mice were euthanized by incision of 
 the caudal vena cava under anesthesia ( intraperitoneal injection of pentobarbital ) . after 
 euthanasia , each liver was dissected and divided into 2 pieces for analysis of enzyme 
 activity and gene expression . the livers for gene expression analysis were treated with rnalater ( qiagen , hilden , 
 germany ) and stored at 80c . the livers used for enzyme analysis were homogenized in 4 vol . 
 of 0.15 m kcl solution containing 1 mm edta . the homogenates were centrifuged for 30 min at 
 9,000 g , and the supernatant fractions were centrifuged at 105,000 g for 60 min to obtain 
 microsomes . the dissolved 
 microsomes were activated by incubation with 0.01% sodium cholate for 30 min at 0c . analysis of ugt activity : udp - glucuronosyltransferase activity toward bpa 
 substrates was assayed in a 400 l volume containing 0.1 m tris - hcl buffer 
 ( ph7.4 ) , 5 mm mgcl2 , 0.2 mm bpa , 3 mm udp glucuronic acid and 10 mg microsomal 
 protein . the mixture was incubated at 37c for 30 min 
 and then boiled for 5 min to stop the reaction . after boiling , the mixture was centrifuged 
 at 12,000 g , and the supernatant was filtered using a disposable disc filter ( hlc - disk3 , 
 kanto chemical co. ) . each supernatant was then analyzed using a dual pump ( dp-8020 ) hplc 
 system ( tosoh corp . , tokyo , japan ) with a fluorescent photometer ( fs-8020 ) and a column oven 
 ( co-8020 ) . samples were separated at 40c using a unison uk - c18 reverse - phase column ( 2.0 mm 
 i.d .  150 mm ; imtakt corp . , kyoto , japan ) at a flow rate of 1.0 ml / min 
 under a linear gradient of solution a ( methanol / water=24/76 vol / vol with 10 mm ammonium 
 acetate , ph=7.0 ) and solution b ( methanol ) for 20 min . the results were recorded using lc-8020 integration 
 software ( tosoh corp . ) ; the elution peaks of bpa and its conjugates were noted , and the 
 concentrations were compared with the standards . the detection limit of assay was 0.2 
 m . to identify the metabolites , lc - esi - tof / ms analysis was performed using a lct premier xe 
 ( waters corp . ) . the eluted peaks in the hplc analysis described above were collected and 
 infused at a flow rate of 0.2 ml / min into the lc - esi - tof / ms using ic-3100 
 syringe driver ( matsuura corp . , analysis of gene expression : total rna for rt - pcr was isolated using an 
 rneasy mini kit and rnase - free dnase 1 ( qiagen ) from tissues treated with rnalater ( qiagen ) , 
 according to the manufacturer s instructions . total rna was converted to cdna using a 
 revertra ace ( toyobo ) reverse transcriptase and oligo dt primers ( toyobo ) , according to the 
 manufacturer s instructions . quantitative analysis of ugt2b1 mrna was 
 normalized to glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) . pcr was 
 performed with the following primers accordingly to a previous report : ugt2b1 sense primer , 5- agatgatggggaaggcagat-3 , 
 and ugt2b1 antisense primer , 5- gcaagagcagaagcaactac-3 ; 
 ugt1a6 sense primer , 5-cctcagtgaacgcggacacgac-3 , and 
 ugt1a6 antisense primer , 5- ttcctgtactctcttagaggagcca-3 , gapdh 
 sense primer , 5-ttcaacggcacagtcaag-3 , and gapdh antisense primer , 
 5-cacacccatcacaaacat-3. the nucleotide sequences for these primers were designed using dna 
 sequences obtained from genbank . pcr amplification was performed as follows : denaturation at 
 94c for 30 sec , annealing at 64c for 30 sec and extension at 72c for 1 min for 35 cycles . ugt2b1 and ugt1a6 were amplified from diluted cdna and 
 used for quantitative rt - pcr analysis . the amplified fragments for each gene were cloned 
 into the pstblue-1 acceptor vector ( merck millipore ) and subsequently transformed into 
 escherichia coli dh5. plasmid vectors with each of the appropriate genes 
 were prepared and quantified using a spectrophotometer . a standard curve for each gene was 
 produced using 100 or 10-fold serial dilutions of the template 
 ( 1010 copies ) . the reaction was performed using a quantitect sybr 
 green pcr kit ( qiagen ) and iq5/myiq single - color ( bio - rad laboratories ) , following the 
 manufacturer s instructions . the correlation coefficient for ugt1a6 , ugt2b1 and gapdh were 
 1.000 , 0.981 and 0.993 , respectively . the amplification efficiency for ugt1a6 , ugt2b1 and 
 gapdh were 87.3% , 80.1% and 115% , respectively . the copy number of each gene expressed in 
 the liver was calculated from their respective standard curves and normalized to that of 
 gapdh ( fig . 2.quantitative analysis of ugt2b1 and ugt1a6 gene expression by real - time rt - pcr . quantitative analysis data of ugt2b1 ( a ) and ugt1a6 ( b ) mrna expression in the liver 
 of male offspring ( white ) , female offspring ( light gray ) and mother ( dark gray ) are 
 shown from gestation day ( gd ) 18.5 to post - natal day 21 . the 
 significant differences of expression in each stage were shown in an asterisk 
 ( p<0.05 ) . ) . quantitative analysis of ugt2b1 and ugt1a6 gene expression by real - time rt - pcr . quantitative analysis data of ugt2b1 ( a ) and ugt1a6 ( b ) mrna expression in the liver 
 of male offspring ( white ) , female offspring ( light gray ) and mother ( dark gray ) are 
 shown from gestation day ( gd ) 18.5 to post - natal day 21 . the 
 significant differences of expression in each stage were shown in an asterisk 
 ( p<0.05 ) . statistical analysis : all data were presented as the mean  s.e . , and the 
 means were compared by use of anova , with the p value of 0.05 as the level 
 of significance . in this study , ugt activity toward bpa , bpf and bpaf was examined in hepatic microsomes 
 purified from perinatal mice and mothers ( fig . 1.ugt activity toward bpaf , bpf and bpa in mouse liver during perinatal development . ugt activity in male offspring ( mo : a , d and g ) , female offspring ( fo : b , e and h ) and 
 mothers ( m : c , f and i ) toward bpaf ( a , b and c ) , bpf ( d , e and f ) and bpa ( g , h and 
 i ) are shown from gestation day ( gd ) 18.5 to post - natal day 21 . ugt activity values 
 are shown as means  s.e . in each chemical , the significant differences of ugt 
 activity of male and female for mother in each stage were shown in an asterisk 
 ( p<0.05 ) . the significant differences of ugt activity toward 
 bpaf compared with both bpa and bpf in each stage were shown in # 
 ( p<0.05 ) . ) . intriguingly , ugt 
 activity toward bpaf was extremely low compared with bpa and bpf in fetuses , pups and dams 
 ( fig . 1a1c ) . furthermore , the activity was 
 significantly low compared with mother at developmental stages ( gd18.5-pd7 in fig . 1a1c ) , and activity did not 
 increase to levels similar to that of the dams in newborn mice ( fig . ugt activity toward bpaf , bpf and bpa in mouse liver during perinatal development . ugt activity in male offspring ( mo : a , d and g ) , female offspring ( fo : b , e and h ) and 
 mothers ( m : c , f and i ) toward bpaf ( a , b and c ) , bpf ( d , e and f ) and bpa ( g , h and 
 i ) are shown from gestation day ( gd ) 18.5 to post - natal day 21 . ugt activity values 
 are shown as means  s.e . in each chemical , the significant differences of ugt 
 activity of male and female for mother in each stage were shown in an asterisk 
 ( p<0.05 ) . the significant differences of ugt activity toward 
 bpaf compared with both bpa and bpf in each stage were shown in # 
 ( p<0.05 ) . in comparison , hepatic ugt activity toward bpf was significantly weak in fetuses and 
 newborn pups , and increased to levels similar to those for bpa after birth ( fig . furthermore , similar levels of ugt 
 activity toward bpf and bpa were observed during pup development and in dams . hepatic ugt2b1 expression was statistically significantly low during the perinatal period 
 ( less than 5.5% of dams ) and increased from pd3 ( about half level of dams ) , becoming similar 
 to levels observed in the dams by pd 7 ( fig . on the other hand , hepatic ugt1a6 expression was slightly low ( about half level of dams ) 
 during the perinatal period ( fig . accordingly , it was thought that bpf was mainly metabolized by ugt2b1 , like bpa . ugt facilitates the excretion of endogenous and xenobiotic compounds , such as bilirubin , 
 steroids and environmental pollutants , as water - soluble conjugates . ugt1a1 mainly catalyzes the glucuronidation of bilirubin , and the 
 absence of ugt1a1 causes crigler - najjar syndrome . ugt1a6 and ugt1a7 catalyze the glucuronidation of similar substrates , such as 
 planar phenols and benzo[a]pyrene . ugt2b1 
 catalyzes the glucuronidation of a number of xenobiotics and endobiotics ( steroids ) , such as 
 estrogen and morphine . previously , we reported that bpa is highly glucuronidated primarily by ugt2b1 in the rat 
 liver [ 10 , 11 , 
 36 ] . we also demonstrated that bpa - ga is 
 transferred into the fetus by uterine perfusion , and we detected not only bpa - ga but also 
 deconjugated bpa in the fetus and amniotic fluid . therefore , elucidation of the pharmacokinetics of bisphenols during the 
 perinatal period is essential to recognizing the mechanism of adverse effects in 
 offspring . it was reported that bpaf is mainly metabolized to a glucuronide - conjugated metabolite in 
 sd rats and that human metabolism is primarily mediated by ugt2b7 . ugt2b7 is known to be an ortholog of the rodent ugt2b1 gene   and is weakly expressed in human fetal liver . ugt2b1 was also weakly expressed in rodent fetuses 
 and newborn pups [ 23 , 26 ] . bpaf has been shown 
 to induce estrogenic effects in vitro [ 17 , 18 ] , exhibiting agonist activity toward 
 er and antagonist activity toward er . bpaf has 
 also been reported to be an agonist of the human pregnane x receptor . exposure to bpaf disrupts hormonal balance and gene expression in 
 the hypothalamus - pituitary - gonad axis and liver , and also decreases egg fertilization in 
 offspring of zebrafish [ 30 , 35 ] and rats , possibly due to 
 sperm deterioration . as a result , it is thought that bpaf has high toxicity , despite being considered a safe 
 replacement for bpa . this may be attributable , at least in part , to the extremely low 
 perinatal bpaf metabolism observed here . in comparison , hepatic ugt activity toward bpf was very weak in fetuses and newborn pups , 
 and increased to levels similar to those for bpa after birth ( fig . accordingly , it was thought that bpf was mainly 
 metabolized by ugt2b1 , like bpa . further works are needed to clarify kinetic properties by 
 using isolated ugt isoenzyme mainly metabolizing bpaf . some reports have shown that bpf is 
 easily metabolized to bpf - glucuronide and bpf - sulfate in hepg2 and human primary hepatocytes 
 [ 2 , 6 ] . it was 
 also shown that bpf is efficiently absorbed through the oral route and distributed to the 
 reproductive tract in pregnant rats , and its residue passed the placental barrier at the 
 late stage of gestation . both animal [ 5 , 32 ] and human 
 [ 7 , 37 ] 
 studies have demonstrated that bpa and bpa - ga are detected in fetuses and amniotic fluid , 
 suggesting the presence of a placental transfer mechanism . we also demonstrated that bpa - ga 
 is transferred into the fetus following uterine perfusion and that bpa - ga and deconjugated 
 bpa are detected in the fetus and amniotic fluid . these reports suggest that maternal and perinatal metabolism , as well as the 
 pharmacokinetics of placenta transfer to the fetus , should be taken into consideration when 
 assessing adverse effects in offspring caused by bisphenol analogs . , we will have to 
 confirm whether bpas induce the ugt activity or not . in summary , we demonstrated that hepatic ugt activity toward bpaf was very low compared 
 with bpa and bpf in fetuses , pups and dams . ugt activity toward bpf was also found to be 
 weak in perinatal offspring , in a manner similar to bpa . further studies are required to 
 clarify the pharmacokinetics of bisphenol analog transfer from mother to fetuses and pups to 
 elucidate the mechanisms of adverse effects on subsequent generations .
bisphenol a ( bpa ) is a widely used industrial chemical that disrupts endocrine function . 
 bpa is an endocrine disrupting chemical ( edc ) that has been demonstrated to affect 
 reproductive organ development , brain development , metabolic disease and post - natal 
 behavior . 
 accordingly , bisphenol analogs , bisphenol f ( bpf , bis ( 4-hydroxyphenyl ) methane ) 
 and bisphenol af ( bpaf , 4,4-hexafluoroisopropylidene ) diphenol ) are used as replacements 
 for bpa . 
 bpa is mainly metabolized by udp - glucuronosyltransferase ( ugt ) , ugt2b1 , but this 
 effective metabolizing system is weak in the fetus . in the present study 
 , we demonstrated 
 that hepatic ugt activity toward bpaf was very weak , in comparison with bpa and bpf , in 
 the fetus , pups and dams . 
 conversely , hepatic ugt activity toward bpf was very weak in the 
 fetus and newborn pups , and was increased to the same level as bpa post - partum . in 
 conclusion , bpaf possibly tends to accumulate in the fetus , because of weak metabolism 
 during the perinatal period , suggesting that the metabolism of individual bisphenol 
 analogs requires assessment to properly gauge their risks .