Quantification of the tissue distribution and accumulation of the neonicotinoid pesticide clothianidin and its metabolites in maternal and fetal mice.

Neonicotinoids (NNs) are commonly used pesticides that have a selective agonistic action on insect nicotinic acetylcholine receptors. Recent evidence has shown that NNs have adverse effects in the next generation of mammals, but it remains unclear how NNs transferred from dams to fetuses are distributed and accumulated in fetal tissues. Here, we aimed to clarify the tissue distribution and accumulation properties of the NN clothianidin (CLO) and its 6 metabolites in 7 tissues and blood in both dams and fetuses of mice administered CLO for a single day or for 9 consecutive days. The results showed that the total concentrations of CLO-related compounds in the brain and kidney were higher in fetuses than in dams, whereas in the liver, heart, and blood they were lower in fetuses. The multi-day administration increased the total levels in heart and blood only in the fetuses of the single administration group. In addition, dimethyl metabolites of CLO showed fetus/dam ratios >1 in some tissues, suggesting that fetuses have higher accumulation property and are thus at higher risks of exposure to CLO-related compounds than dams. These findings revealed differences in the tissue-specific distribution patterns of CLO and its metabolites between dams and fetuses, providing new insights into the assessment of the developmental toxicity of NNs.

Sex-specific behavioral effects of acute exposure to the neonicotinoid clothianidin in mice.

Although neonicotinoids are among the major classes of pesticides that affect mammalian nervous systems, little is known about sex differences in their effects. This study aimed to examine whether the neurobehavioral effects of a neonicotinoid, clothianidin (CLO), differed between sexes. Male and female C57BL/6N mice were orally administered CLO (5 or 50 mg/kg) at or below the chronic no-observed-adverse-effect-level (NOAEL) and subjected to behavioral tests of emotional and learning functions. Changes in neuroactivity in several brain regions and the concentrations of CLO and its metabolites in blood and urine were measured. Acute CLO exposure caused sex-related behavioral effects; decreases in locomotor activities and elevation of anxiety-like behaviors were more apparent in males than in females. In addition, male-specific impairment of short- and long-term learning memory by CLO exposure was observed in both the novel recognition test and the Barnes maze test. Male-dominant increases in the number of c-fos positive cells were observed in the paraventricular thalamic nucleus in the thalamus and in the dentate gyrus in the hippocampus, which are related to the stress response and learning function, respectively. The concentrations of CLO and most metabolites in blood and urine were higher in males. These results support the notion that male mice are more vulnerable than females to the neurobehavioral effects of CLO and provide novel insights into the risk assessment of neonicotinoids in mammalian neuronal function.

Aging-related changes in the sensitivity of behavioral effects of the neonicotinoid pesticide clothianidin in male mice.

Neonicotinoids, which act as agonists of the nicotinic acetylcholine receptors of insects, are widely used pesticides worldwide. Although epidemiological studies revealed that the detection amounts of neonicotinoids in urine are higher in the elderly population than other age-groups, there is no available information regarding the risks of neonicotinoids to older mammals. This study was aimed to investigate aging-related differences in the behavioral effects of the neonicotinoid pesticide clothianidin (CLO). We acutely administered a sub-NOAEL level (5 mg/kg) of CLO to adult (12-week-old) and aging (90-week-old) mice and conducted four behavioral tests focusing on the emotional behavior. In addition, we measured the concentrations of CLO and its metabolites in blood, brain and urine. There were age-related changes in most parameters in all behavioral tests, and CLO significantly decreased the locomotor activity in the open field test and elevated plus-maze test in the aging group, but not in the adult group. The concentrations of most CLO and its metabolites were significantly higher in the blood and brain and were slightly lower in the urine in the aging group compared to the adult group. These findings should contribute to our understanding of age-related differences in the adverse effects of neonicotinoids in mammals.

Establishment of an organ culture system to induce Sertoli cell differentiation from undifferentiated mouse gonads.

Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Müllerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.

Quantitative elucidation of maternal-to-fetal transfer of neonicotinoid pesticide clothianidin and its metabolites in mice.

Neonicotinoids (NNs), a widely used class of systemic pesticides, are regarded as exhibiting selective toxicity in insects. However, NNs are suspected of exerting adverse effects on mammals as well, including humans. To date, only adult male animal models have been subjected to general toxicity studies of NNs; fetuses have yet to be considered in this context. Here, we focused on the NN clothianidin (CLO) for the first quantitative LC-MS/MS analysis of maternal-to-fetal transfer and residual property of once-daily (single or multiple days), orally administered CLO and its metabolites in mice. The results revealed the presence of CLO and its five metabolites at approximately the same respective blood levels in both dams and fetuses. In the dams, CLO showed a peak value 1 h after administration, after which levels rapidly decreased at 3 and 6 h. In the fetuses of each group, levels of CLO were almost the same as those observed in the corresponding dams. The present results clearly demonstrated rapid passage of CLO through the placental barrier. However, metabolite-dependent differences observed in blood pharmacokinetics and residual levels. This is the first quantitative demonstration of the presence of CLO and its metabolites in fetal mouse blood.

Immunotoxicity evaluation by subchronic oral administration of clothianidin in Sprague-Dawley rats.

Neonicotinoid pesticides (NNs) act as agonists on nicotinic acetylcholine receptors (nAChRs) of insects, and there have been concerns about the effects of NNs on the health of mammals. Since nAChRs are expressed in immune cells, it is possible that NNs disturb the immune system. However, few reports have examined the immunotoxicity of clothianidin (CLO), a widely-used NN. Here, we report the effects of CLO on immune organs and type IV allergic reactions in ear auricles. We orally administered CLO at 0, 30 and 300 mg/kg/day (CLO-0, 30 and 300) to Sprague-Dawley rats for 28 days. The effects were evaluated by organ and body weights, histopathology, and immunohistochemistry (TCRαß, CD4, CD8, CD11b, CD68, CD103). In addition, some cecal contents were subjected to preliminary gut microbiota analysis, because microbiota contribute to host homeostasis, including the immunity. Our results showed loose stool, suppression of body weight gain, significant changes in organ weights (thymus: decreased; liver: increased) and changes of the gut microbiota in the CLO-300 group. There were no obvious histopathological changes in immune organs. Granulomas of the ear auricles were found in one rat of each of the CLO-30 and 300 groups, but CLO had no apparent effect on the thickness or immunohistochemistry in the ear auricles. We present new evidence that CLO affects the thymus and intestine, and might enhance the local inflammatory response. These findings should contribute to the appropriate evaluation of the safety of NNs in the future.

Evaluation of the Water Content and Skin Permeability of Active Pharmaceutical Ingredients in Ketoprofen Poultice Formulations Removed from Their Airtight Containers and Left at Room Temperature.

The poultice formulation is a patch containing a large amount of water. It is known that the water contained in the adhesive polymer layer (ADPL) of poultice affects the cooling sensation and skin permeability of the active pharmaceutical ingredient (API). In this study, we evaluated the relationship between the water content in a ketoprofen poultice formulation and the amount of time the poultice was left out at room temperature after removal from the airtight container, as well as the influence of the decreasing water content on the skin permeability of the API. After removing the poultice from the container for 1 h, the mass of the ADPL decreased by approximately 40%. When the near-infrared (NIR) spectrum of the ADPL of poultice was measured, the peaks reflecting the hydroxyl group were attenuated depending on the time left out at room temperature. It is suggested that the changes in the mass and NIR spectrum of the ADPL are caused by the change in the water content. Moreover, when the permeability of API was evaluated on hairless mouse skin, the cumulative skin permeation amount and flux decreased, while the lag time was prolonged as the time left out increased. These results suggest that the skin permeability of the API is impaired by water evaporation and that maintaining the water in the ADPL in poultice is very important from not only the viewpoint of cooling sensation, tackiness and moisturizing but also the skin permeability of the API.

Effect of ß-agonist on the dexamethasone-induced expression of aromatase by the human monocyte cells.

Emerging evidence suggests that sex steroids are important for human skin health. In particular, estrogen improves skin thickness, elasticity and moisture of older women. The major source of circulating estrogen is the ovary; however, local estrogen synthesis and secretion have important roles in, for example, bone metabolism and breast cancer development. We hypothesized that infiltrated peripheral monocytes are one of the sources of estrogen in skin tissues. We also hypothesized that, during atopic dermatitis under stress, a decline in the hypothalamus-pituitary-adrenal axis (HPA) and facilitation of the (hypothalamus)-sympathetic-adrenomedullary system (SAM) attenuates estrogen secretion from monocytes. Based on this hypothesis, we tested aromatase expression in the human peripheral monocyte-derived cell line THP-1 in response to the synthetic glucocorticoid dexamethasone (Dex), the synthetic ß-agonist isoproterenol (Iso) and the ß-antagonist propranolol (Pro). Dex mimics glucocorticoid secreted during excitation of the HPA, and Iso mimics catecholamine secreted during excitation of the SAM. We found that aromatase activity and the CYP19A1 gene transcript were both upregulated in THP-1 cells in the presence of Dex. Addition of Iso induced their downregulation and further addition of Pro rescued aromatase expression. These results may suggest that attenuation of estrogen secretion from peripheral monocytes could be a part of the pathology of stress-caused deterioration of atopic dermatitis. Further examination using an in vitro human skin model including THP-1 cells might be a valuable tool for investigating the therapeutic efficacy and mechanism of estrogen treatment for skin health.

Evidence for expression of 11ß-hydroxysteroid dehydrogenase type3 (HSD11B3/HSD11B1L) in neonatal pig testis.

11ß-hydroxysteroid dehydrogenase (HSD11B) catalyzes the interconversion between active and inactive glucocorticoid, and is known to exist as two distinct isozymes: HSD11B1 and HSD11B2. A third HSD11B isozyme, HSD11B1L (SCDR10b), has recently been identified. Human HSD11B1L, which was characterized as a unidirectional NADP(+)-dependent cortisol dehydrogenase, appears to be specifically expressed in the brain. We previously reported that HSD11B1 and abundant HSD11B2 isozymes are expressed in neonatal pig testis and the Km for cortisol of NADP(+)-dependent dehydrogenase activity of testicular microsomes obviously differs from the same activity catalyzed by HSD11B1 from pig liver microsomes. Therefore, we hypothesized that the neonatal pig testis also expresses the third type of HSD11B isozyme, and we herein examined further evidence regarding the expression of HSD11B1L. (1) The inhibitory effects of gossypol and glycyrrhetinic acid on pig testicular microsomal NADP(+)-dependent cortisol dehydrogenase activity was clearly different from that of pig liver microsomes. (2) A highly conserved human HSD11B1L sequence was observed by RT-PCR in a pig testicular cDNA library. (3) mRNA, which contains the amplified sequence, was evaluated by real-time PCR and was most strongly expressed in pig brain, and at almost the same levels in the kidney as in the testis, but at lower levels in the liver. Based on these results, neonatal pig testis appears to express glycyrrhetinic acid-resistant HSD11B1L as a third HSD11B isozyme, and it may play a physiologically important role in cooperation with the abundantly expressed HSD11B2 isozyme in order to prevent Leydig cell apoptosis or GC-mediated suppression of testosterone production induced by high concentrations of activated GC in neonatal pig testis.

Effects of bisphenol A on the expression of cytochrome P450 aromatase (CYP19) in human fetal osteoblastic and granulosa cell-like cell lines.

The effects of bisphenol A (BPA), an endocrine disruptor, on aromatase (CYP19) expression in human osteoblastic (SV-HFO) and ovarian granulosa-like (KGN) cell lines were examined. CYP19 enzyme activity was suppressed in the presence of BPA in a dose-dependent fashion in both cell lines. CYP19 gene transcript expression, as well as activities of promoter I.4 in SV-HFO and promoter II in KGN, was down-regulated by BPA, suggesting that BPA affects CYP19 at the gene-expression level. These data and the previous finding that BPA induced the down-regulation of promoter I.1 activity within the human placental cell line suggest that there may be a conserved signaling pathway that down-regulates CYP19 expression in response to BPA in both cell lines. Additionally, differences between promoter I.4 and II suggest that there may be cell- and promoter-specific down-regulating mechanisms downstream from the actions of BPA.

Quantitative analysis of UGT2B28 mRNA expression by real-time RT-PCR and application to human tissue distribution study.

We recently established a method for quantitative determination of human catalytically active UDP-glucuronosyltransferases (UGTs) other than UGT2A1 and UGT2B28 by real-time reverse transcription-polymerase chain reaction (RT-PCR), and applied the method to an exhaustive analysis of localization in various human tissues. We report here an additional quantitative determination method targeting UGT2B28. To date, there have been no reports on the distribution of UGT2B28 mRNA expression in human tissues based on quantitative determination. Human UGT2B28 was clearly detected in the breast and adipose tissue. UGT2B28 expression in the breast was comparatively low, about 1.6% of GAPDH mRNA levels, and was less than 5% of normalized (against GAPDH) UGT2B7 and 2B10 mRNA expression levels in the liver. Although the UGT2B28 has 97% identity with UGT2B11 at the cDNA sequence level, the primers constructed for UGT2B28 did not detect UGT2B11. In addition, significant expression of UGT2B11 was detected in the liver, breast and kidney, and was clearly different from the distribution of UGT2B28. Therefore, we conclude that the real-time RT-PCR method established here is very specific for human UGT2B28 isozyme.

Quantitative determination and tissue distribution of human 11ß-hydroxysteroid dehydrogenase, hexose-6-phosphate dehydrogenase, glucose-6-phosphate transporter, glucocorticoid receptor and mineralocorticoid receptor mRNAs.

BACKGROUND: Glucocorticoid (GC) concentrations in peripheral tissues are precisely regulated by 11ß-hydroxysteroid dehydrogenase (HSD) isozymes. When considering the physiological effects of GC in various tissues, quantitative determination of these isozymes and other components involved in corticosteroid signaling is important and informative. We thus performed comprehensive determination of the expression of these mRNAs in a wide range of human tissues. METHODS: An absolute comparison of mRNA expression of human 11ß-HSD isozymes, hexose-6-phosphate dehydrogenase (H6PDH), glucose-6-phosphate transporter (G6PT), glucocorticoid receptors (GRs), and mineralocorticoid receptor (MR) was performed by real-time RT-PCR. RESULTS: Human 11ß-HSD type 1 mRNA was strongly expressed in the liver and placenta at comparatively high levels. H6PDH was expressed at low copy number, and comparatively high expression was observed in the kidney, testis, and ovary. G6PT expression was ubiquitous, but marked expression was observed in the liver, kidney, small intestine, and colon. GRα was also ubiquitously expressed at relatively high levels, which were approximately 10-fold higher than those of MR, whereas GRß levels were below the detection limit in all tissues. 11ß-HSD type 2 was predominantly expressed in the kidney, small intestine and colon; however, copy numbers of these transcripts showed a nearly identical pattern to type 1. MR was observed in various tissues examined, but was not fully correlated to the distribution of 11ß-HSD type 2. CONCLUSIONS: The present quantitative results were partially consistent with previous studies. This quantification method can thus provide valuable information for understanding the physiological effects and physiological roles of glucocorticoid in humans.

Sequence and expression of 11beta-hydroxysteroid dehydrogenase type 1 cDNA cloned from pig testis.

Pig 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11beta-HSD type 1, and the sequence of 11beta-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region. The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11beta-HSD type 1.

Mono-(2-ethylhexyl) phthalate induces NR4A subfamily and GIOT-1 gene expression, and suppresses CYP19 expression in human granulosa-like tumor cell line KGN.

The mechanism for transcriptional suppression of CYP19 by mono-ethylhexyl phthalate (MEHP) in a human ovarian granulosa cell line (KGN) was investigated. It is known that the CYP19 gene transcript in KGN cells predominantly includes exon PII among the 11 alternate exon I sequences. MEHP was found to significantly suppress Forskolin (FSK)-induced CYP19 gene transcription, CYP19 promoter II activity and CYP19 enzyme activity in a dose-dependent manner. Promoter assays using 5'-deleted promoter II reporter constructs suggested that the region important for responsiveness to MEHP exposure includes a putative CRE-like sequence and an SF-1 (NR5A1)/LRH-1 (NR5A2) binding sequence. Meanwhile, MEHP exposure rapidly and transiently induced nuclear receptor 4A (NR4A) mRNA, and gradually and continuously induced gonadotropin-inducible ovarian transcription factor-1 (GIOT-1; ZNF461) mRNA in KGN cells. The ectopic expression of NR4As and GIOT-1 suppressed promoter II activity, while among the NR4As expressed, only Nur77 (NR4A1) secondarily induced GIOT-1 mRNA expression. Based on these results, we believe that induction of the Nur77-GIOT-1 system by MEHP is involved in the transcriptional suppression of the CYP19 gene, and GIOT-1 may attenuate the promoter II activity due to suppression of SF-1 and/or LRH-1 transactivation in KGN cells.

Determination of mRNA expression of human UDP-glucuronosyltransferases and application for localization in various human tissues by real-time reverse transcriptase-polymerase chain reaction.

An exhaustive real-time reverse transcriptase-polymerase chain reaction (PCR) quantification method was used to determine 15 of the catalytically active human UDP-glucuronosyltransferase (UGT) isoforms (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17). The specific primers for respective human UGTs were developed for differential determination. The cDNA derived from the 1A7 isoform was detected in the esophagus, the 1A8 and 1A10 isoforms were detected in the small intestine, and all other isoforms were detected in at least the liver by PCR. In all cases, single bands of the expected size on the agarose gel were confirmed to correspond with the predicted UGT isoform sequences. Each calibration curve showed linearity between the PCR crossing point and the calibrator copy number. The correlation coefficients were greater than 0.9957 with high reproducibility. This exhaustive measurement method was applied to UGT expression in 23 human tissue types. UGT was mostly expressed in the alimentary system and liver. We were surprised to find that extremely high expression in the liver was found for UGT2B4 and UGT2B15, which had, respectively, 8.98 and 4.38 times greater expression than UGT2B7 in the liver. In addition, even though expressed at low levels, several UGT isoforms were expressed in steroidogenic tissues, such as the breast, prostate, heart, and adrenal. Therefore, this quantification method may provide valuable information about the medical efficacy or pharmacokinetic characteristics of a wide variety of UGT-metabolized drugs.

Contribution of UDP-glucuronosyltransferase 1A1 and 1A8 to morphine-6-glucuronidation and its kinetic properties.

The metabolic conversion of morphine to morphine-6-glucuronide (M6G) seems to play a significant role in mediation of the clinical effect of morphine because of the superior analgesic effect of M6G. Therefore, it would be of great interest to clarify the specificity of morphine-6-glucuronidation by UDP glucuronosyltransferase (UGT) isozymes. We investigated the specificity of morphine-6-glucuronidation catalyzed by recombinant human UGT isozymes in microsomes from baculovirus-infected insect cells. The morphine glucuronidation activity of recombinant human UGT isozymes incubated with morphine and UDP-glucuronic acid was determined by high-performance liquid chromatography with a fluorescence detector. Not only UGT2B7, which is well known to catalyze morphine-6-glucuronidation, but also UGT1A1 and 1A8 effectively catalyzed morphine-6-glucuronidation at relatively low morphine concentrations (<100 muM). The kinetics of both isozymes at the low substrate concentrations showed hyperbolic Michaelis-Menten kinetics. However, as the morphine concentration approached 100 muM, morphine-6-glucuronidation activity gradually decreased, and the kinetics closely resembled substrate inhibition Michaelis-Menten kinetic behavior. The K(m) values were 67.9 and 68.1 muM and the K(si) values were 218.9 and 88.0 muM for UGT1A1 and 1A8, respectively. These kinetics are basically different from that of morphine-6-glucuronidation by UGT2B7, which suggested biphasic Michaelis-Menten kinetic behavior. Furthermore, to estimate the contribution of these UGT isozymes in M6G formation in vivo, the expression levels of UGT1A1 and 1A8 mRNA in human liver and intestine were determined by reverse transcription real-time polymerase chain reaction. The results strongly suggest that UGT1A1 and UGT1A8 are isozymes involved in morphine-6-glucuronidation in vivo, as is UGT2B7 in humans.

Expression in E. coli and tissue distribution of the human homologue of the mouse Ke 6 gene, 17beta-hydroxysteroid dehydrogenase type 8.

Expression of the human Ke 6 gene, 17beta-hydroxysteroid dehydrogenase type 8, in E. coli and the substrate specificity of the expressed protein were examined. The tissue distribution of mRNA expression of the human Ke 6 gene was also studied using real-time PCR. Human Ke 6 gene was expressed as an enzymatically-active His-tag fusion protein, whose molecular weight was estimated to be 32.5 kDa by SDS-polyacrylamide gel electrophoresis. Expressed human Ke 6 gene effectively catalyzed the conversion of estradiol into estrone. Testosterone, 5alpha-dihydrotestosterone, and 5-androstene-3beta,17beta-diol were also catalyzed into the corresponding 17-ketosteroid at 2.4-5.9% that of estradiol oxidation. Furthermore, expressed enzyme catalyzed the reduction of estrone to estradiol, but the rate was a mere 2.3%. Human Ke 6 gene mRNA was expressed in the various tissues examined, such as brain, cerebellum, heart, lung, kidney, liver, small intestine, ovary, testis, adrenals, placenta, prostate, and stomach. Expression of human Ke 6 gene mRNA was especially abundant in prostate, placenta, and kidney. The levels in prostate and placenta were higher than that in kidney, where it is known to be expressed in large quantities.

Mono-(2-ethylhexyl) phthalate (MEHP) induces nuclear receptor 4A subfamily in NCI-H295R cells: a possible mechanism of aromatase suppression by MEHP.

Phthalate esters are widely used as plasticizers for polyvinylchloride and are suspected of functioning as endocrine disrupters. Di-(2-ethylhexyl) phthalate (DEHP), the most important phthalate ester in commercial use, has been reported to act as a rodent reproductive toxicant. In the present study, we investigated the effects of phthalate esters on aromatase (CYP19) activity and on its gene expression in a human adrenocortical carcinoma cell line, NCI-H295R. Mono-(2-ethylhexyl) phthalate (MEHP), a principle metabolite of DEHP, dose-dependently suppressed aromatase activity and its transcription level. Furthermore, MEHP rapidly and transiently induced transcription of the genes which encode nuclear receptor 4A subfamily members (Nur77, Nurr1 and NOR-1), and up-regulated Nur77 promoter activation and Nur77 protein expression in the cells. MEHP-induced Nur77 transcription was inhibited by bisindolylmaleimide I (protein kinase C inhibitor) and wortmannin (phosphoinositide 3-kinase inhibitor). Finally, ectopic expression of Nur77 markedly suppressed forskolin-induced transcriptional activation of promoters I.3 and II of the CYP19 gene. These results suggest that the suppression of aromatase activity and its transcription level by MEHP exposure to NCI-H295R cells was regulated through the rapid and transient expression of Nur77 gene.

Sequence of pig 17beta-hydroxysteroid dehydrogenase Type3 cDNA and its expression in mammalian cells.

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) Type3 is an NADPH-dependent membrane-bound enzyme that is specifically expressed in testis and catalyzes the conversion of androstenedione to testosterone. To date, the sequence of Type3 enzymes has been clarified in humans, mice and rats; however, the sequence of the pig enzyme remains unknown. In this study, we determined the cDNA sequence of pig testicular 17beta-HSD Type3. PCR primers for partial pig testicular 17beta-HSD Type3 were designed from rat and human enzyme consensus sequences. Full-length cDNA was obtained by 3'- and 5'-RACE based on partial PCR products. The cDNA coding region was 933 bp in length, which is the same as the human enzyme, and shared 84.7% sequence identity with the human cDNA coding region. The monomer was estimated to have a molecular weight of 34,855 and to contain 310 amino acid residues. The predicted pig amino acid sequence showed 81.9, 75.5 and 72.9% sequence identity with the human, rat and mouse sequences, respectively. To elucidate 17beta-HSD Type3 activity, the expression vector pCMV/pig17beta-HSD3 was established and transfected into human embryo kidney 293 cells. Subsequently, 17beta-HSD activity (androstenedione conversion to testosterone) was strongly detected in cell lysates.

Inhibitory effects of some possible endocrine-disrupting chemicals on the isozymes of human 11beta-hydroxysteroid dehydrogenase and expression of their mRNA in gonads and adrenal glands.

The in vitro inhibitory effects of 23 compounds designated as possible endocrine-disrupting chemicals, such as alkylphenols, phthalate esters, phytoestrogens, organotins, and pesticides, on the enzyme activity of the isozymes of human 11beta-HSD, type 1 and 2, were examined. Human liver microsomal 11beta-HSD type 1 was inhibited by 4-nonylphenol (4-NP) and diethylhexyl adipate (IC50 = 49.3 and 49.4 mM, respectively). Further, human kidney microsomal 11beta-HSD type 2 was inhibited by triphenyltin, tributyltin, 4-t-octylphenol, 4-NP, 4-n-octylphenol, 4-n-nonylphenol, and dicyclohexyl phthalate (IC50 = 3.3, 16.5, 8.9, 20.3, 23.5, 26.2 and 46.5 microM, respectively). The other chemicals studied did not significantly inhibit the enzyme at concentrations lower than 50 microM. The expression of mRNA of the isozymes in gonads, accessory genital glands, and adrenal glands were investigated by RT-PCR. 11beta-HSD type 1 was detected in human testis, ovary, and prostate. The level in the adrenal gland(s) was similar to that in the liver, which is known to be expressed in large quantities. In contrast, except for the prostate, only small amounts of the 11beta-HSD type 2 isozyme were detected in these human tissues compared to kidney.