Causes of Sex Differences in Serum Cholesterol and Triglyceride Levels in Meishan Pigs.

To clarify the causes of sex differences (male < female) in the serum total cholesterol (TCHO) and triglyceride (TG) levels in Meishan pigs, we examined the sex differences in mRNA levels of key hepatic enzymes involved in the biosynthesis/metabolism of cholesterol and TG using real-time RT-PCR. There were no sex differences in mRNA levels of 3-hydroxy-3-methylglutaryl-CoA reductase and CYP51A1 for cholesterol biosynthesis, or of the rate-limiting enzyme CYP7A1 for bile acid synthesis from cholesterol. By contrast, sex differences (male < female) were observed in mRNA levels of glycerol-3-phosphate acyltransferase 1 (GPAT1), a rate-limiting enzyme for TG biosynthesis. However, the sex differences in mRNA levels of carnitine palmitoyltransferase 1A (CPT1A) and acyl-CoA dehydrogenase long chain (ACADL), key enzymes for the oxidation of the fatty acids that are structural components of TG, were the opposite (male > female). Castration of male pigs led to an increase in the mRNA level of GPAT1 and decreases in those of CPT1A and ACADL. Furthermore, testosterone propionate (TP)-treatment of castrated males and intact females restored and changed, respectively, these mRNA levels to those of intact males. Notably, castration and TP-treatment increased and decreased, respectively, serum and hepatic TG levels. These findings suggest that sex differences in the serum and hepatic TG levels in Meishan pigs are closely correlated with differences in testosterone-associated mRNA expression levels of the key enzymes (GPAT1, CPT1A, and ACADL) involved in the TG biosynthesis process, although no causes of sex differences in serum and hepatic TCHO levels could be found.

Breed Differences in Serum Testosterone Levels of Male Pigs Are Highly Correlated with Differences in Testicular mRNA Levels of Enzymes and Proteins Involved in Testosterone Biosynthesis Process.

To clarify the causes of breed differences in serum testosterone levels of male pigs, which affect the mRNA expression of drug metabolizing enzymes and drug transporters in the liver and kidney, we focused on testicular enzymes and proteins involved in testosterone biosynthesis process and comparatively examined their mRNA levels by real time RT-PCR among low serum testosterone-type Landrace pigs and high serum testosterone-type Meishan and Landrace/Meishan-crossbreed (LM and ML) pigs. Testicular mRNA levels of the enzymes (3-hydroxy-3-methylglutaryl-CoA synthase 1 and 3-hydroxy-3-methylglutaryl-CoA reductase) and proteins (low density lipoprotein receptor and scavenger receptor class B member 1) affecting intracellular levels of cholesterol, a precursor of testosterone, were 2-5-fold higher in Meishan, LM and ML pigs than in Landrace pigs. Likewise, the mRNA levels of steroidogenic acute regulatory protein, which imports cholesterol to the inner mitochondrial membrane, and of testosterone biosynthesis enzymes (CYP11A1 and CYP17A1) were over 10-fold and approximately 3-fold higher, respectively, in Meishan, LM and ML pigs than in Landrace pigs. Furthermore, positive correlations between those mRNA levels and serum testosterone levels were observed. Despite large breed differences in testicular mRNA levels described above, no significant breed differences in intratesticular testosterone levels were observed. The present findings strongly suggest that breed differences in serum testosterone levels of male pigs are probably, at least in part, caused by differences in testicular mRNA levels of enzymes and proteins involved in testosterone biosynthesis process and by differences in the levels of testosterone released from testes.

Effect of Growth Hormone Receptor Deficiency on Androgen-Associated Gene Expression of Hepatic Drug Metabolizing Enzymes and Drug Transporters in Pigs.

Growth hormone receptor (GHR)-deficient pigs were generated using the CRISPR/Cas9 system to investigate the involvement of GHR-mediated growth hormone (GH) signaling in androgen-associated gene expression of hepatic drug metabolizing enzymes (DMEs) and drug transporters. We initially confirmed that no wild-type GHR mRNA was present in GHR-/- (GHR-KO) pigs; in addition, as previously reported, those pigs exhibited decreases in body weight and serum insulin-like growth factor-1 concentration and an increase in serum GH concentration compared with the levels in GHR-/+ and GHR+/+ pigs with a wild-type GHR mRNA. The real-time RT-PCR results on the mRNA levels of hepatic DMEs and drug transporters in the GHR-KO pigs and the pigs with a wild-type GHR mRNA revealed that, among the examined hepatic DMEs, the mRNA levels of CYP1A2, CYP2A19, sulfotransferase (SULT) 1A1, and SULT2A1 were higher in GHR-KO pigs than in the pigs with a wild-type GHR mRNA, whereas the opposite trend was observed for the mRNA level of uridine 5'-diphospho-glucuronosyltransferase 1A6. No such significant differences in the mRNA levels of three hepatic drug transporters including multidrug resistance protein 1 were observed. In addition, the mRNA level of hepatic cut-like homeobox 2 (CUX2), which is expressed in an androgen-dependent manner and associated with the hepatic mRNA expression of several DMEs, was significantly decreased in GHR-KO pigs. The present findings strongly suggest that not only serum androgen but also GHR-mediated GH signaling contributes to the mRNA expression of several DMEs and CUX2, but not transporters, in the pig liver.

Androgen-Dependent Expression of CUX2 mRNA in the Pig Liver Is Associated with That of Drug Metabolizing Enzymes and Drug Transporters.

We previously identified androgen-dependent sex differences in the mRNA expression of drug metabolizing enzymes (DMEs), including CYPs, sulfotransferases and uridine 5'-diphospho-glucuronosyltransferases, and drug transporters in the pig liver and kidney. To elucidate the mechanism for such sex differences in pigs, we herein focused on the key regulators cut-like homeobox 2 (Cux2), B-cell lymphoma 6 (Bcl6), and signal transducer and activator of transcription 5b (Stat5b), which are reported to be responsible for the sex-biased gene expression of Cyps in the mouse liver. We used real-time RT-PCR to examine androgen-dependent sex differences in the mRNA levels of these regulators in the liver and kidney basically using Meishan and Landrace pigs. Significant sex differences (male > female) in the level of CUX2 mRNA were detected in the liver of both breeds, and levels were significantly decreased in males by castration and increased in castrated males and intact females by administering testosterone propionate. No such clear androgen-dependent sex differences in hepatic BCL6 or STAT5B mRNA expression were observed in either breed. In the kidney, androgen-dependent gene expression of these regulators was not observed. In the liver, CUX2 mRNA expression closely correlated with that of DMEs and drug transporters, which were previously shown to have androgen-dependent expression. Together, these findings demonstrate that hepatic CUX2 mRNA is expressed in an androgen-dependent manner, and strongly suggest that CUX2 plays a key role in the androgen-dependent gene expression of hepatic DMEs and drug transporters.

Sex, Organ, and Breed Differences in the mRNA Expression of Drug Transporters in the Liver and Kidney of Pigs.

Domestic pigs are attractive as an animal model for humans because of their anatomical and physiological similarities to humans. In this study, sex, organ, and breed differences in the mRNA expression of drug transporters such as breast cancer resistance protein (BCRP), multidrug resistance protein 1 (MDR1), multidrug resistance associated protein 2 (MRP2), organic anion porting polypeptide 1B3 (OATP1B3), organic anion transporters (OAT1, OAT2, and OAT3), and organic cation transporters (OCT1 and OCT2) were examined by RT-PCR in the liver and kidney of 5-month-old Meishan and Landrace pigs. No sex differences in the amount of BCRP mRNA were observed in both breeds. In Meishan pigs, sex differences (male < female) in the mRNA amounts of MDR1, OATP1B3, and OCT1 were observed in the liver. Similarly, sex differences in the mRNA amounts of MRP2, OAT1, OAT2, OAT3, and OCT2 were observed in the kidney of Meishan pigs: male > female for MRP2, OAT3, and OCT2, and male < female for OAT1 and OAT2. However, no such sex differences were observed in Landrace pigs. In addition, regardless of breed, hepatic OAT1, OAT3, and OCT2 mRNAs and renal OATP1B3 mRNA were not detected. Thus, organ and breed differences in the expression of drug transporters suggest the existence of genetically controlled organ-selective factors. Furthermore, additional experiments in castrated and/or testosterone propionate-treated pigs strongly suggested that sex and breed differences in the gene expression of drug transporters, especially hepatic OCT1 and renal OAT1, were primarily due to the difference in serum testosterone concentration.

Androgen-Dependent Differences in the Amounts of CYP mRNAs in the Pig Kidney.

We previously reported androgen-dependent sex and breed differences in the amounts of mRNAs of CYP isoforms in the pig liver. To clarify whether there are such sex and breed differences in the kidney, we examined the amounts of several CYP mRNAs in the kidney using both sexes of 5-month-old Landrace, Meishan and/or their crossbred F1 (LM and ML) pigs. Significant sex differences in the amounts of several CYP mRNAs were found: male < female for CYP2A19 and CYP3A29; and male > female for CYP4A24/25 in all the breeds. Sex differences in the amount of CYP2B22 mRNA (male < female) and in CYP2C33 and CYP2C49 mRNAs (male > female) were also observed in all the breeds except Landrace pigs. Furthermore, a significant sex difference (male < female) in CYP3A46 mRNA was only found in LM and ML pigs. No significant sex differences were found in either Landrace or Meishan pigs for CYP1A1, CYP1A2 and CYP4B1 mRNAs. The amounts of CYP2C33 and CYP4A24/25 mRNAs in males were higher in Meishan pigs than in Landrace pigs. Additional experiments using pigs treated by castration and/or testosterone propionate indicated that sex and breed differences in the amounts of those CYP mRNAs were, at least in part, dependent on the levels of serum testosterone. Furthermore, the effects of androgen on the amounts of CYP mRNAs in the kidney did not necessarily correlate with those in the liver, suggesting that there is a tissue-selective factor responsible for the androgen-related expression of CYP genes.

The Differential Selectivity of Aryl Hydrocarbon Receptor (AHR) Agonists towards AHR-Dependent Suppression of Mammosphere Formation and Gene Transcription in Human Breast Cancer Cells.

We had previously reported that treatment with the aryl hydrocarbon receptor (AHR) agonist ß-naphthoflavone (ßNF) suppressed mammosphere formation derived from cancer stem cells in human breast cancer MCF-7 cells (Cancer Lett., 317, 2012, Zhao et al.). Here, using several AHR agonists, we have investigated the association of this suppression with the classical ability to induce AHR-mediated gene transcription in the xenobiotic response element (XRE). The mammosphere formation assays were performed using wild-type and AHR-knockout MCF-7 cells in the presence of AHR agonists including 3-methylcholanthrene (3MC), benzo[a]pyrene (BaP), 7,12-dimethylbenz[a]anthracene (DMBA), 6-formylindolo[3,2-b]carbazole (FICZ), indirubin, indole-3-carbinol (I3C), indole-3-acetic acid (IAA), and kynurenine (KYN), followed by the XRE-reporter gene assays of the agonists. We showed that treatments with 3MC, BaP, and DMBA strongly suppressed mammosphere formation of the stem cells in an AHR-dependent manner, while other agonists showed weaker suppression. In reporter gene assays, the strength or duration of AHR/XRE-mediated gene transcription was found to be dependent on the agonist. Although strong transcriptional activation was observed with 3MC, FICZ, indirubin, I3C, IAA, or KYN after 6 h of treatment, only weak activation was seen with BaP or DMBA. While transcriptional activation was sustained or increased at 24 h with 3MC, BaP, or DMBA, appreciable reduction was observed with the other agonists. In conclusions, the results demonstrated that the suppressive effects of AHR agonists on mammosphere formation do not necessarily correlate with their abilities to induce AHR-mediated gene transcription. Hence, different AHR functions may be differentially induced in an agonist-dependent manner.

Kanechlor 500-mediated changes in serum and hepatic thyroxine levels primarily occur in a transthyretin-unrelated manner.

The effects of Kanechlor-500 (KC500) on the levels of serum total thyroxine (T4 ) and hepatic T4 in wild-type C57BL/6 (WT) and its transthyretin (TTR)-deficient (TTR-null) mice were comparatively examined. Four days after a single intraperitoneal injection with KC500 (100 mg/kg body weight), serum total T4 levels were significantly decreased in both WT and TTR-null mice. The KC500 pretreatment also promoted serum [125 I]T4 clearance in both strains of mice administrated with [125 I]T4 , and the promotion of serum [125 I]T4 clearance in WT mice occurred without inhibition of the [125 I]T4 -TTR complex formation. Furthermore, the KC500 pretreatment led to significant increases in liver weight, steady-state distribution volume of [125 I]T4 , hepatic accumulation level of [125 I]T4 , and concentration ratio of the liver to serum in both strains of mice. The present findings indicate that the KC500-mediated decrease in serum T4 level occurs in a TTR-unrelated manner and further suggest that KC500-promoted T4 accumulation in the liver occurs through the development of liver hypertrophy and the promotion of T4 transportation from serum to liver.

Possible mechanism for the polychlorinated bipheny-linduced liver-selective accumulation of thyroxine in rats.

We have previously reported that decrease in level of serum thyroxine T4 by Kanechor 500 (KC500) in rats would occur through the increase in hepatic T4 accumulation rather than the increase in hepatic T4-glucuronyl transferase activity. In the present study, to understand the mechanism underlying the KC500-mediated increase in hepatic T4 accumulation, we examined the relationship between the KC500-mediated changes in hepatic T4 accumulation and the expression levels of mRNAs of hepatic transporters including T4 transporters. [125I]T4 was intravenously injected into KC500-pretreated and control (KC500-untreated) Wistar rats, and [125I]T4 uptake levels of liver parenchymal cells were comparatively examined. The amount of [125I]T4 uptake by hepatic cells increased in a time-dependent manner up to 96 hr after KC500 treatment. Following KC500 treatment, a time-dependent increase in the mRNA level of hepatic T4 influx transporter LAT1 was observed up to 96 hr later, while a significant increase in hepatic T4 influx transporter Oatp2 mRNA occurred only at 96 hr later. No KC500-mediated increases in the mRNAs of other hepatic transporters (Oatp1, Oatp3, Oatp4, Ntcp, LAT2, and Mrp2) were observed at any timepoints, although the mRNA expression of the T4 conjugate(s) efflux transporter Mrp3 significantly increased in a time-dependent manner 24-96 hr following KC500 treatment. The present findings suggest that KC500-mediated increase in hepatic T4 accumulation occurs, at least in part, through the increase in the expression of hepatic T4-transporters, such as LAT1 and Oatp2.

Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of food-flavouring substances uniquely used in Japan that belong to the class of aliphatic primary alcohols, aldehydes, carboxylic acids, acetals and esters containing additional oxygenated functional groups.

We performed a safety evaluation using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) of the following four flavouring substances that belong to the class of 'aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups' and are uniquely used in Japan: butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate. Although no genotoxicity study data were found in the published literature, none of the four substances had chemical structural alerts predicting genotoxicity. All four substances were categorised as class I by using Cramer's classification. The estimated daily intake of each of the four substances was determined to be 0.007-2.9 µg/person/day by using the maximised survey-derived intake method and based on the annual production data in Japan in 2001, 2005 and 2010, and was determined to be 0.250-600.0 µg/person/day by using the single-portion exposure technique and based on average-use levels in standard portion sizes of flavoured foods. Both of these estimated daily intake ranges were below the threshold of toxicological concern for class I substances, which is 1800 µg/person/day. Although no information from in vitro and in vivo toxicity studies for the four substances was available, these substances were judged to raise no safety concerns at the current levels of intake.

Aryl hydrocarbon receptor activation and CYP1A induction by cooked food-derived carcinogenic heterocyclic amines in human HepG2 cell lines.

The ability of nine cooked food-derived heterocyclic aromatic amines (HCAs), such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methylpyrido[12-a:3',2'-d]imidazole (Glu-P-1), 2-amino-pyrido[12-a:3',2'-d]imidazole hydrochloride (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP), to activate human aryl hydrocarbon receptor (hAhR) was examined using a HepG2-A10 cell line, which has previously established from human hepatocarcinoma-derived HepG2 cells for use in hAhR-based luciferase reporter gene assays. Trp-P-1, Trp-P-2, AαC, MeAαC, IQ and MeIQx showed a definite ability to induce not only luciferase (hAhR activation) in HepG2-A10 cells but also cytochrome P450 (CYP)1A1/1A2 mRNAs in HepG2 cells, while such the ability of Glu-P-1, Glu-P-2, and PhIP was very low. In addition, all the HCAs examined, especially MeAαC and MeIQx, had a definite capacity for inhibiting the activity of ethoxyresorfin O-deethylase (CYP1As, especially CYP1A1). The present findings demonstrate that all the HCAs examined have the ability to activate hAhR and its target genes, and further confirm that these HCAs become good substrates for human CYP1A subfamily enzyme(s).

Sex differences in constitutive mRNA levels of CYP2B22, CYP2C33, CYP2C49, CYP3A22, CYP3A29 and CYP3A46 in the pig liver: Comparison between Meishan and Landrace pigs.

Breed and sex differences in hepatic mRNA levels of cytochrome P450 (CYP) isoforms (CYP2B22, CYP2C33, CYP2C49, CYP3A22, CYP3A29 and CYP3A46) were examined in 5-month-old Meishan, Landrace, and their crossbred F1 (LM and ML) pigs. Serum testosterone levels in male Meishan, LM, and ML pigs were 2.5-3.5-fold higher than in Landrace pigs. CYP3A46 mRNA was breed-specifically detected only in Landrace, LM, and ML pigs. In Meishan, LM, and ML pigs only, male-predominant expressions of CYP2B22, CYP2C33, CYP2C49 and CYP3A29 mRNAs were observed; CYP3A22 mRNA expression showed the opposite pattern. Male-dominant mRNA expression was also observed in LM and ML pigs for CYP3A46. The sex differences in CYP mRNA levels in Meishan pigs disappeared when males were castrated and were restored by testosterone propionate (TP) administration to the castrated males. In Landrace pigs, TP administration to castrated males and intact females significantly increased the levels of CYP2B22, CYP2C33, and CYP3A46 mRNAs. Immature (1-month-old) pigs showed no breed or sex differences in CYP mRNA expressions. The results demonstrated that androgen is an important determinant of sex-associated expression of several CYPs and suggested that breed differences in sex-associated expression could be caused by differences in serum androgen level and by other genetic traits.

The Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of acetal food flavouring substances uniquely used in Japan.

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on five acetal flavouring substances uniquely used in Japan: acetaldehyde 2,3-butanediol acetal, acetoin dimethyl acetal, hexanal dibutyl acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal. As no genotoxicity study data were available in the literature, all five substances had no chemical structural alerts predicting genotoxicity. Using Cramer's classification, acetoin dimethyl acetal and hexanal dibutyl acetal were categorised as class I, and acetaldehyde 2,3-butanediol acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal as class III. The estimated daily intakes for all five substances were within the range of 1.45-6.53 µg/person/day using the method of maximised survey-derived intake based on the annual production data in Japan from 2001, 2005, 2008 and 2010, and 156-720 µg/person/day using the single-portion exposure technique (SPET), based on the average use levels in standard portion sizes of flavoured foods. The daily intakes of the two class I substances were below the threshold of toxicological concern (TTC) - 1800 µg/person/day. The daily intakes of the three class III substances exceeded the TTC (90 µg/person/day). Two of these, acetaldehyde 2,3-butanediol acetal and hexanal glyceryl acetal, were expected to be metabolised into endogenous products after ingestion. For 4-methyl-2-pentanone propyleneglycol acetal, one of its metabolites was not expected to be metabolised into endogenous products. However, its daily intake level, based on the estimated intake calculated by the SPET method, was about 1/15 000th of the no observed effect level. It was thus concluded that all five substances raised no safety concerns when used for flavouring foods at the currently estimated intake levels. While no information on in vitro and in vivo toxicity for all five substances was available, their metabolites were judged as raising no safety concerns at the current levels of intake.

Usefulness of urinary kidney injury molecule-1 (Kim-1) as a biomarker for cisplatin-induced sub-chronic kidney injury.

We explored biomarkers suitable for monitoring sub-chronic kidney injury using the three rat models of cisplatin (CDDP)-induced kidney injury, which were designed to extend the current knowledge beyond the sub-acute exposure period. In the pilot study, a single intravenous administration of 1.5 mg kg(-1) CDDP to rats was confirmed to result in no histopathological changes. Subsequently, CDDP was intravenously administered to rats at a dose of 1.5 mg kg(-1) for 4 days at 24-h intervals (Experimental model 1) and for up to 10 weeks at weekly intervals (Experimental models 2 and 3), and the changes in blood and urine components, such as recently recommended urinary biomarkers (Kim-1, clusterin and so on) and traditional blood biomarkers (blood urea nitrogen and serum creatinine), were examined together with the histopathological changes in renal tissues during the development of the kidney injury in each model. In these experimental models, a significant increase in urinary Kim-1 was observed prior to the histopathological changes in renal tissues, and these changes were retained after the adverse histopathological changes. Significant changes in all of the other urinary biomarkers examined occurred along with the histopathological changes. In addition, the increase in urinary Kim-1 after weekly treatment with CDDP for 4 weeks was reduced in a time-dependent manner after cessation of the drug. The present findings indicate that urinary Kim-1 is the most useful biomarker for CDDP-induced rat sub-chronic kidney injury among the biomarkers examined.

Upregulation of N-methyl-D-aspartate receptor subunits and c-Fos expressing genes in PC12D cells by nobiletin.

The N-methyl-D-aspartate (NMDA) receptor plays a key role in learning and memory. Our recent studies have shown that nobiletin from citrus peels activates the cAMP response element-binding protein (CREB) signaling pathway and ameliorates NMDA receptor antagonist-induced learning impairment by activating extracellular signal-regulated kinase. For the first time, we have shown that nobiletin significantly upregulated mRNA expression of the NMDA receptor subunits NR1, NR2A, and NR2B in PC12D cells. Furthermore, c-Fos mRNA expression also increased due to the action of nobiletin. Our results indicate that nobiletin modulates the expression of essential genes for learning and memory by activating the CREB signaling pathway, and suggest that this action mechanism of nobiletin plays a crucial role in improving NMDA receptor antagonist-induced learning impairment in model animals with dementia.

Involvement of hepatic IL-1 in the strain-dependent sex differences in serum total cholesterol levels in rats.

The strain and sex differences in serum total cholesterol (TC) levels were examined in F344 and Sprague-Dawley (SD) rats. A sex difference (male

The JFFMA assessment of flavoring substances structurally related to menthol and uniquely used in Japan.

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on four flavoring substances structurally related to menthol (L-menthyl 2-methylbutyrate, DL-menthyl octanoate, DL-menthyl palmitate, and DL-menthyl stearate) uniquely used in Japan. While no genotoxicity study data were available in the literature, all four substances had no chemical structural alerts predictive of genotoxicity. Moreover, they all four are esters consisting of menthol and simple carboxylic acids that were assumed to be immediately hydrolyzed after ingestion and metabolized into innocuous substances for excretion. As menthol and carboxylic acids have no known genotoxicity, it was judged that the JECFA procedure could be applied to these four substances. According to Cramer's classification, these substances were categorized as class I based on their chemical structures. The estimated daily intakes for all four substances were within the range of 1.54-4.71 µg/person/day and 60-1250 µg/person/day, using the methods of Maximized Survey-Derived Intake and Single Portion Exposure Technique, respectively, based on the annual usage data of 2001, 2005, and 2010 in Japan. As the daily intakes of these substances were below the threshold of concern applied to class I substances viz., 1800 µg/person/day, it was concluded that all four substances raise no safety concerns when used for flavoring foods under the currently estimated intake levels.

Sex differences in the constitutive gene expression of sulfotransferases and UDP-glucuronosyltransferases in the pig liver: androgen-mediated regulation.

Using two breeds (Meishan and Landrace) of pigs and their crossbred offspring (ML, Meishan × Landrace; LM, Landrace × Meishan), of which males have genetically different serum androgen levels, we examined whether or not serum androgen plays a crucial role in the constitutive gene expression of hepatic sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs). Real-time RT-PCR analyses showed that in Meishan, ML, and LM pigs, SULT1A1 and SULT2A1 mRNA levels were lower in males having high levels of serum androgen (>38 ng/ml) than in females, whereas those of UGT1A1, UGT1A6, and UGT2B31 were just the opposite. In Landrace pigs having low levels of serum androgen (<22 ng/ml), no such sex differences in expression levels were observed. Moreover, castration of male Meishan pigs altered the gene expression patterns of SULTs and UGTs to female levels. Testosterone-treatment to the castrated males and intact females of either pig breed resulted in decreased SULT1A1 and SULT2A1 and increased UGT1A1, UGT1A6, and UGT2B31 mRNA levels. These findings demonstrate that androgen is one of the physiological factors that determine sexual dimorphism on the constitutive gene expression of SULTs and UGTs in the pig liver.

3,3',4,4'-Tetrachlorobiphenyl-mediated decrease of serum thyroxine level in C57BL/6 and DBA/2 mice occurs mainly through enhanced accumulation of thyroxine in the liver.

A single intraperitoneal injection (50 mg/kg) of 3,3',4,4'-tetrachlorobiphenyl (CB77), a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-type polychlorinated biphenyl, led to significant decreases in the levels of serum total thyroxine (T4) and free T4 without increase in the level of serum thyroid-stimulating hormone at 7 d later in both TCDD-sensitive C57BL/6 and TCDD-resistant DBA/2 mice. When [(125)I]T4 was injected into the mice 7 d after treatment with CB77, the levels of biliary [(125)I]T4 and [(125)I]T4-glucuronide increased 90 to 120 min post injection in C57BL/6 mice, but not in DBA/2 mice, compared with levels in the corresponding control mice. In contrast, in both strains of mice, the CB77-pretreatment led to similar changes in the levels of the [(125)I]T4 bound to the serum transthyretin, albumin, and thyroxine-binding globulin. Consequently, treatment with CB77 promoted the clearance of [(125)I]T4 from the serum and further raised the steady-state volumes of distribution of [(125)I]T4, the concentration ratio (Kp value) of the liver to the serum, and the distribution of [(125)I]T4 in the liver in both strains of mice. The present findings indicate that in mice, the CB77-mediated decrease in the serum T4 level occurs through enhanced accumulation of hepatic T4 rather than through increased activity of hepatic thyroxine-uridine 5'-diphosphate-glucuronosyltransferase(s).

6-demethoxynobiletin, a nobiletin-analog citrus flavonoid, enhances extracellular signal-regulated kinase phosphorylation in PC12D cells.

We previously demonstrated that nobiletin, a polymethoxylated flavone isolated from citrus peels, has the potential to improve cognitive dysfunction in patients with Alzheimer's disease (AD). Recent studies suggest that the generation of intraneuronal amyloid-beta (Aß) oligomers is an early event in the pathogenesis of AD. Aß oligomers cause deficits in the regulation of the extracellular signal-regulated kinase (ERK) signaling which is critical for consolidation of the memory. Our previous studies revealed that nobiletin activated ERK signaling and subsequent cyclic AMP response element-dependent transcription. In this study, the effects of five nobiletin analogs, 6-demethoxynobiletin, tangeretin, 5-demethylnobiletin, sinensetin, and 6-demethoxytangeretin, isolated from citrus peels were assessed on ERK phosphorylation in PC12D cells, and the structure-activity relationships were examined. PC12D cells were treated with nobiletin or its analogs, and the cell extracts were analyzed by Western blotting using an antibody specific to phosphorylated ERK. 6-Demethoxynobiletin markedly enhanced ERK phosphorylation in a concentration-dependent manner. These results may be useful in developing drugs and functional foods using citrus peels for the treatment of dementia including AD.