[Anti-diabetic effect of ethanol extract of Cyclolepis genistoides D. Don (Palo azul), made in Paraguay].

Extract of Cyclolepis genistoides D. Don (vernacular name Palo azul; Palo) are traditionally consumed in the Republic of Paraguay in South America for the treatment of diabetes and kidney disease, and is sold in Japan as dietary supplement. This study aimed to elucidate the mechanism of anti-diabetes activity of Palo, especially focused on insulin resistance. Palo promoted adipocytes differentiation and regulated adipokine profiles in 3T3-L1 adipocytes by modulation of PPARγ, a major regulator of adipose differentiation. Human adipocyte showed almost similar profile with 3T3-L1 against Palo treatment. Furthermore, Palo treatment (250 or 1000 mg/kg) was performed with C57BL/6J mice for 14 weeks, being fed high-fat-diet (HFD60) simultaneously. Palo 250 mg/kg exhibited a tendency to decrease subcutaneous adipose volume along with increase of PPARγ and its target, adiponectin mRNA expression. In addition, as the other insulin targeted cell, effect on muscle differentiation was examined. Palo increased differentiation of C2C12 mouse muscle myoblasts by increase of IGF-1, myogenin, and myosine heavy chain (MHC) as well as 5'-AMP-activated protein kinase (AMPK) activation. Palo subsequently promoted myotube formation under differentiation condition. From the above, it was clarified that Palo acts variously on the differentiation and maturation of both adipocytes and muscle cells, and from the viewpoint of the regulatory mechanism for adipocytes, PPARγ-inducing action was shown to be a mechanism that acts across species.

Ethanol extract of Cyclolepis genistoides D. Don (palo azul) induces formation of myotubes, which involves differentiation of C2C12 myoblast cells.

In this study, we examined the cell differentiation effect of an ethanol extract of Cyclolepis genistoides D. Don, a herbaceous perennial belonging to the family Asteraceae (vernacular name: palo azul). Palo azul has numerous physiological effects that contribute to the prevention of metabolic syndromes, although the mechanism remains unclear. We previously suggested that palo azul has antidiabetic activity via an adipose differentiation effect. Here, we focused on whether palo azul promoted the differentiation of myoblasts. The mouse muscle myoblast cell line C2C12 was cultured and differentiated using horse serum with or without an ethanol extract of palo azul (12.5-200 µg/mL). Quantitative real-time polymerase chain reaction was performed to evaluate differentiation markers, including insulin-like growth factor-1 and myogenin. To evaluate myotube formation, myosin heavy-chain (MHC) expression and localization were detected by immunohistochemistry. Palo azul increased the expression of the differentiation markers. Furthermore, immunohistochemistry analysis revealed increased formation of MHC myotubes after palo azul treatment along with increased diameter and fusion indices of the myotubes. The expression level of MHC was also increased. In conclusion, palo azul may increase muscle mass in the body and improve insulin resistance conditions by facilitating the formation of myotubes by promoting myocyte differentiation.

Protective effect of young green barley leaf (Hordeum vulgare L.) on restraint stress-induced decrease in hippocampal brain-derived neurotrophic factor in mice.

BACKGROUND: Many health experts support the hypothesis that stressful lifestyles are the leading cause of illness, like depression. Therefore, from the standpoint of preventive medicine, it is important to reduce stress. Young green barley leaves are a good natural source of vitamins and minerals, and their juice is widely consumed as a functional food for health reasons in Japan. This study investigated the protective effect of young green barley leaves for stress control. MATERIALS AND METHODS: ICR outbred mice were exposed to 3-h sessions of restraint stress. Young green barley leaves (400 and 1,000 mg/kg) were administered orally 1 h before the sessions for 5 days. To analyze voluntary behavior, wheel-running activity was monitored during the dark period. Brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) expression in the whole hippocampus was measured by real-time quantitative polymerase chain reaction. RESULTS: Restraint stress resulted in a significant decrease in voluntary wheel-running behavior, but this decrease was ameliorated by the administration of young green barley leaves. The leaves also enhanced the decreased levels of BDNF mRNA induced by restraint stress; in particular, a significant protective effect was shown in the exon IV variant as compared to vehicle control mice. CONCLUSION: The findings suggest that young green barley leaves have potent anti-stress properties, as evidenced by preventing decreases in the levels of voluntary wheel-running activity and hippocampal BDNF mRNA in response to restraint stress. Our findings support the possibility that supplementation with young green barley leaves might be beneficial for preventing stress-related psychiatric disorders like depression.

Polychlorinated biphenyl levels in the blood of Japanese individuals ranging from infants to over 80 years of age.

Individuals' exposure to various persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs), and its adverse health effects have been a cause of concern. We measured blood PCB concentrations from samples taken from 507 Japanese individuals ranging from infants to those over 80 years of age. The blood PCB levels increased with age for both male (Spearman's r = 0.69, p < 0.001) and female (Spearman's r = 0.70, p < 0.001) participants. Adult men and nulliparous women showed similar increases with age. However, the PCB levels of multiparous women were lower than those of nulliparous women in their thirties (p = 0.005), probably because the PCBs were transferred from the mothers to their children during pregnancy and lactation. Among infants (<2 years of age), some had as high levels of accumulated PCB levels as those in adults >30 years of age. In some cases, the PCB levels were over 0.8 ng/g wet weight, similar to levels observed in adults over 50 years of age. In the future, it will be necessary to do research on the health of the children who are exposed by high concentration level of POPs.

Sex differences in stress reactivity of hippocampal BDNF in mice are associated with the female preponderance of decreased locomotor activity in response to restraint stress.

The incidence and prevalence of depression is higher in women than in men, but the cause of this sex discrepancy remains unknown. Brain-derived neurotrophic factor (BDNF) is a key protein for maintaining neuronal integrity. The purpose of this study was to investigate the female preponderance in behavioral responsivity to restraint stress focusing on the stress reactivity of BDNF in the hippocampus. Male and female ICR mice were exposed to a 3-h session of restraint stress. Plasma corticosterone was measured by high-performance liquid chromatography. BDNF mRNA expression in the whole hippocampus was measured by quantitative real-time reverse transcription-polymerase chain reaction. Wheel-running activity was monitored during the dark period. In response to restraint stress, the increase in levels of serum corticosterone was higher in female than in male mice. Restraint stress resulted in decreased voluntary wheel-running behavior that was greater in female than male animals. In addition to these sex differences in stress reactivity, we found a significant sex difference in BDNF levels in the hippocampus of restraint-stressed mice; total BDNF levels significantly decreased in female mice, but not in male mice in response to the stress. Furthermore, BDNF exon I and IV mRNA expression also showed the same tendency. These data indicate that the reduction in levels of voluntary wheel-running activity in response to stress can be significantly influenced by sex. Moreover, our findings suggest a link between the sex differences in this behavioral response to stress and differential stress reactivity in the production of BDNF in the hippocampus.

Cyclolepis genistoides D. Don (palo azul) promotes differentiation of adipocytes and regulates adipokine expression.

Cyclolepis genistoides D. Don is a herbaceous perennial belonging to the family Asteraceae, and its vernacular name is "palo azul" (palo). Palo has been reported to exhibit many physiological effects that contribute to the prevention of metabolic syndromes, although its mechanism is unclear. Among palo's various activities, we investigated the hypothesis that palo promotes adipocytes differentiation and regulates adipokine profiles in 3T3-L1 adipocytes by modulation of peroxisome proliferator-activated receptor (PPAR) γ, a major regulator of adipose differentiation. 3T3-L1 adipocytes were cultured and differentiated in Dulbecco modified Eagle medium with 50 to 200 µg/mL palo for 7 days or were cultured with palo without differentiation protocol for 14 days. Palo down-regulated the expression of 2 types of expressed/secreted adipokines, leptin and resistin, in a concentration-dependent manner. Under a nondifferentiated condition, palo promoted the accumulation of lipid droplets in cells. Real-time polymerase chain reaction and luciferase reporter assay showed that palo up-regulated expression and transcriptional activity of PPARγ. Furthermore, palo increased the expression of insulin-sensitizing adipokine, adiponectin, which is a directly target of PPARγ, both at the messenger RNA level and at the protein level. In summary, palo demonstrated the potential to improve insulin resistance by promoting adipocyte differentiation via PPARγ activation. Results suggest an increase in adiponectin secretion and a decrease in insulin-resistant factors such as leptin and resistin.

Antidepressant-like effects of young green barley leaf (Hordeum vulgare L.) in the mouse forced swimming test.

BACKGROUND: Young green barley leaf is one of the richest sources of antioxidants and has been widely consumed for health management in Japan. In this study, we examined whether oral administration of young green barley leaf has an antidepressant effect on the forced swimming test in mice. MATERIALS AND METHODS: Mice were individually forced to swim in an open cylindrical container, one hour after oral administration of young green barley leaf (400 or 1000 mg / kg) or imipramine (100 mg / kg). Expression of mRNA for nerve growth factor (NGF), brain-derived neurotrophic factor, and glucocorticoid receptor in the brain was analyzed using real-time quantitative polymerase chain reaction (PCR). RESULTS: There was a significant antidepressant-like effect in the forced swimming test; both 400 and 1000 mg / kg young green barley leaves, as well as the positive control imipramine (100 mg / kg), reduced the immobility duration compared to the vehicle group. The expression of mRNA for NGF detected in the hippocampus immediately after the last swimming test was higher than that in the non-swimming group (Nil). Oral administration of imipramine suppressed this increase to the level of the Nil group. Young green barley leaf (400 and 1000 mg / kg) also showed a moderate decrease in the expression of mRNA for NGF, in a dose-dependent manner. CONCLUSION: Oral administration of young green barley leaf is able to produce an antidepressant-like effect in the forced swimming test. Consequently it is possible that the antidepressant-like effects of the young green barley leaf are, at least in part, mediated by an inhibition of the increase in the hippocampus levels of NGF.

Confirmation of polychlorinated biphenyl (PCB) distribution in the blood and verification of simple quantitative method for PCBs based on specific congeners.

We measured the concentration of each polychlorinated biphenyl (PCB) congener in whole blood, plasma and blood cells, and investigated the distribution of PCBs in human blood using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). The PCB concentrations in plasma and whole blood in terms of lipid concentrations were almost equal, with a correlation coefficient of r=0.972. In the blood, the ratio of PCBs in blood cells to those in plasma was generally about 1:9 and the congener distribution patterns in blood cells and plasma were similar. We performed verification of a simple mass screening method by obtaining information on the main PCB congeners for investigations on human accumulation and exposure. The total concentration of the seven PCB congeners (UNEP-7) proposed to the United Nations Environment Programme (UNEP) by Muir and Morita was about 50% of the total concentration of all PCB congeners, and UNEP-30 was about 80%. The seven main congeners in the blood (MCB-7) showed a value that was about 60%, and MCB-30 showed a value that was about 90%. Determinations with the main congeners in the blood showed a correlation of r=0.990 or more between the main eight congeners (MCB-7 plus #74) and the total PCB concentration for all congeners. The results suggest that, although total PCB concentration can be effectively estimated from the main seven congeners, the main eight congeners would be preferable, and that the use of these congeners in the simple mass screening method would be effective for populations in areas uncontaminated by PCBs.

Polybrominated diphenyl ethers cause oxidative stress in human umbilical vein endothelial cells.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants to prevent combustion in consumer products, such as electronics, construction materials, and textiles and, therefore, have become important commercial substances. PBDEs were also detected in maternal blood, breast milk, umbilical cord blood, and cord tissue, thereby indicating that fetuses were also exposed to PBDEs. The purpose of this study is to identify the effect of PBDEs on human umbilical vein endothelial cells (HUVECs). Cultured HUVECs were exposed to a commercial mixture of penta-BDE (DE71), octa-BDE (DE79), and deca-BDE (DE83). Each gene expression that was altered in DNA microarray was confirmed by real-time reverse transcription-polymerase chain reaction and Western blotting analysis. The results indicated that gene expressions concerning antioxidant system, i.e., thioredoxin family, 24-dehydrocholesterol reductase (DHCR24), and tumor suppressor protein p53, were altered by PBDEs exposure in HUVECs. Moreover, it was demonstrated that thioredoxin-interacting protein (TXNIP) was a target gene in exposure to DE71 and DE79 in HUVECs, by drastically decreasing time-dependent TXNIP expression in HUVECs.

Neonatal exposure to diethylstilbestrol alters expression of DNA methyltransferases and methylation of genomic DNA in the mouse uterus.

Perinatal exposure to diethylstilbestrol (DES) can have numerous adverse effects on the reproductive organs later in life, such as vaginal clear-cell adenocarcinoma. Epigenetic processes including DNA methylation may be involved in the mechanisms. We subcutaneously injected DES to neonatal C57BL/6 mice. At days 5, 14, and 30, expressions of DNA methyltransferases (Dnmts) Dnmt1, Dnmt3a, and Dnmt3b, and transcription factors Sp1 and Sp3 were examined. We also performed restriction landmark genomic scanning (RLGS) to detect aberrant DNA methylation. Real-time RT-PCR revealed that expressions of Dnmt1, Dnmt3b, and Sp3 were decreased at day 5 in DES-treated mice, and that those of Dnmt1, Dnmt3a, and Sp1 were also decreased at day 14. RLGS analysis revealed that 5 genomic loci were demethylated, and 5 other loci were methylated by DES treatment. Two loci were cloned, and differential DNA methylation was quantified. Our results indicated that DES altered the expression levels of Dnmts and DNA methylation.

Perinatal exposure to brominated flame retardants and polychlorinated biphenyls in Japan.

Brominated flame retardants (BFRs) are used to prevent combustion in consumer products. Examples of BFRs are polybrominated diphenyl ethers (PBDEs), tetrabromobisphenol A (TBBPA), and tribromophenol (TBP). These compounds are reported to have adverse effects on human health and endocrine disrupting effects. The purpose of this study was to identify the Japanese perinatal exposure to PBDEs, hydroxylated PBDE metabolites (OH-PBDEs), TBBPA, and TBP compared with polychlorinated biphenyls (PCBs) and hydroxylated PCB metabolites (OH-PCBs). We investigated the concentrations of these compounds in maternal blood, maternal milk, cord blood, and umbilical cords from 16 Japanese mother-infant pairs by HRGC/HRMS. PBDEs were detected in all samples of maternal blood (mean+/-SD; median=25+/-23 pg/g; 18 pg/g wet weight), maternal milk (140+/-220 pg/g; 59 pg/g wet weight), cord blood (4.8+/-6.5 pg/g; 1.6 pg/g wet weight), and umbilical cords (3.1+/-3.1 pg/g; 2.1 pg/g wet weight). The mothers were divided into two groups, a high-concentration group and a low-concentration group. The percentage of BDE-47 showed the greatest difference between the two groups. 6-OH-BDE-47, TBBPA, and TBP were detected in all umbilical cord samples (mean+/-SD; median=8.4+/-8.1 pg/g; 8.0 pg/g, 16+/-5.5 pg/g; 15 pg/g, and 33+/-8.2 pg/g; 32 pg/g wet weight respectively), but not in all maternal blood or cord blood samples. These results indicate that OH-PBDEs, TBBPA, and TBP, in addition to PBDEs, PCBs, and OH-PCBs, pass through the blood-placenta barrier and are retained in the umbilical cord.

Strong correlation between the concentration of dioxins and total PCBs in current Japanese people.

The purpose of this study was to establish an economic and efficient method to screen total PCBs and total dioxins (PCDDs+PCDFs+Co-PCBs) in the highly exposed people in Japan. In this paper, we suggest use of total PCBs in human blood to represent other persistent organic pollutants, especially dioxins. Twenty blood samples were collected from Japanese volunteers. Total PCBs and total dioxins (PCDDs+PCDFs+Co-PCBs) were detected from all twenty blood samples. We carried out detailed analysis of correlation between concentration of total PCBs and each dioxin congener with both measured value and TEQ calculated value. The mean concentration of total PCBs was 250 ng g-fat(-1), and the mean concentration of total dioxins was 37 ng g-fat(-1) or 40 pg TEQ g-fat(-1). Correlations between the total PCBs (ng g-fat(-1)) and the total measured dioxins (ng g-fat(-1)), and between the total PCBs (ng g-fat(-1)) and the total dioxin TEQ calculated value (pg-TEQ g-fat(-1)) were 0.95 and 0.90, respectively. It became clear that the concentrations of total PCBs in human blood is a good indicator of the concentrations of total dioxins in Japan. If a mass screening is conducted on women of reproductive age in order to detect highly exposed women, it is possible that women with the highest contamination may be treated in order to decrease the levels of these chemicals before pregnancy. In conclusion, measurement of total PCBs concentration is useful for exposure assessment of dioxins in human blood.

Polychlorinated biphenyls alter the expression of endothelial nitric oxide synthase mRNA in human umbilical vein endothelial cells.

Polychlorinated biphenyls (PCBs) are a group of persistent pollutants that are detected in maternal serum and umbilical cord, suggesting that fetal exposure also needs to be considered. The effects of dioxin-like PCB congeners 3,3',4,4'-tetrachlorobiphenyl (PCB77) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) and a non-dioxin-like compound 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) on the expression of endothelial nitric oxide synthase (eNOS), known to maintain blood flow to the fetus, in human umbilical vein endothelial cells (HUVECs) were investigated. The mRNA levels of eNOS, aryl hydrocarbon receptor (AhR) and cytochrome P450 (CYP) 1A1 in cells treated with 5 microM PCBs for 24 hours were analysed by real-time RT-PCR. Cells were also treated with alpha-naphthoflavone (alpha NF), an AhR antagonist or ICI 182780, an estrogen receptor (ER) antagonist, one hour prior to PCB exposure, to observe the effects of these receptors on eNOS modulation. Each PCB increased the eNOS mRNA level by 4.5-fold that was markedly inhibited by alphaNF. ERs were also suspected of altering eNOS levels because ICI 182780 treatment resulted in a decrease in the eNOS level. These results suggest that the eNOS mRNA expression increases due to the action of PCBs related to both AhR and ERs in HUVECs, and that maternal PCB exposure could influence fetal circulation.

Reduction of dioxins and polychlorinated biphenyls (PCBs) in human body.

The accumulation of persistent lipophilic organic pollutants like dioxins and PCBs in human body is of great concern since many of these compounds may elicit adverse health effects on humans. To reduce dioxins and PCBs with long half-lives that are absorbed into the human body, we need to work actively to minimize accumulation of dioxins and PCBs taken. Lot of manner has been tested such as foods containing dietary fibers and chlorophyll, lipids (squalane etc) and anion exchange resins. Cholestyramine, a cholesterol lowering agent, was no efficacy in humans. Authors have conducted a pilot study to demonstrate the effect lowering dioxin in human bodies using colestimide. Nine patients on cdestimide for 6 months, showed mean 20% decrease respectively in both dioxin and PCB levels, and the maximum percentage decrease was approximately 40%. From a standpoint to avoid the influence on high-risk group and high-risk life stage other than next generation, the world-wide cooperation for reducing environmental chemicals is greatly appealed.

The contamination levels of organochlorines and the pattern of gene expressions in human umbilical cords from intra-pairs of twins at delivery.

In this study, we examined the contamination levels of organochlorines and the patterns of gene expressions using umbilical cords of twins. The contamination levels of total PCBs, HCB and HCHs of intra-pair of twins were close to each other, but that of DDTs were not so as the others. It shows that the placental or fetal factor may influence the transfer or the accumulation of DDTs. The patterns of gene expressions of intra-pair of twins were similar to each other. It may because the environmental factor (sharing same mother), genetic factor or the both of twins are similar. This study gave us some information to understand the fetal exposure to organochlorines. Moreover, it showed the possibility that the pattern of gene expression in umbilical cord reflected the intra-uterine environment. However, the information from general pattern of the gene expression is limited. Therefore, further investigation is necessary to find new markers not only mRNA but also protein to estimate the effects of fetal exposure of multiple organochlorines.

Neonatal administration of diethylstilbestrol has adverse effects on somatic cells rather than germ cells.

Neonatal administration of diethylstilbestrol (DES) to rodents has adverse effects on spermatogenesis. However, not many studies have been conducted to determine which type of cell - germ or somatic - is the major target of DES. In order to clarify this, we tried reciprocal germ cell transplantation--transplantation of germ cells from DES-treated mice into intact mice and germ cells from normal mice into DES-treated mice. The donor germ cells were tagged with the green fluorescent protein (GFP) gene in order to distinguish the exogenous germ cells from the endogenous cells. Moreover, to obtain a large number of spermatogonia from the testes of adult mice, we performed fractionation by centrifugation with Percoll. Consequently, we found that the germ cells collected from DES-treated mice have differentiated into normal sperms in normal seminiferous tubules. However, in the case of the transplantation of normal germ cells into the seminiferous tubules of DES-treated mice, defective spermatogenesis was observed. In conclusion, DES has adverse effects on the somatic cells that are involved in spermatogenesis rather than the germ cells.

Identification and characterization of novel and unknown mouse epididymis-specific genes by complementary DNA microarray technology.

To examine epididymal function, we attempted to identify highly expressed genes in mouse epididymis using a cDNA microarray containing PCR products amplified from a mouse epididymal cDNA library. We isolated one novel and four known genes-lymphocyte cytosolic protein 1 (Lcp1), complement subcomponents C1r/C1s, Uegf protein, and bone morphogenetic protein and zona pellucida-like domains 1 (Cuzd1), transmembrane epididymal protein 1 (Teddm1), and whey acidic protein 4-disulfide core domain 16 (Wfdc16)-with unknown functions in the epididymis. The novel gene, designated Serpina1f (serine peptidase inhibitor [SERPIN], clade A, member 1f), harbors an open reading frame of 1 233 bp encoding a putative protein of 411 amino acids, including a SERPIN domain. These five genes were predominantly expressed in the epididymis as compared to other organs. In situ hybridization analysis revealed their epididymal region-specific expression patterns. Real-time RT-PCR analysis revealed a significant increase in mRNA expression of these genes around puberty. Castration decreased their expression, except forLcp1. Testosterone (T) restored these reduced expressions, except forTeddm1; however, this restoration was not observed with 17 beta-estradiol (E2). Administration of T and E2 combination recovered the Serpina1f mRNA concentration; this recovery was also observed with T alone. However, the recovery of Cuzd1and Wfdc16mRNA concentrations was inadequate. Neonatal diethylstilbestrol treatment suppressed the Cuzd1, Wfdc16, and Serpina1f mRNA expression in the epididymis of 8-week-old mice; this was not observed with E2. These results suggest that our microarray system can provide a novel insight into the epididymal function on a molecular basis, and the five genes might play important roles in the epididymis.

Neonatal exposure to diethylstilbestrol alters the expression of DNA methyltransferases and methylation of genomic DNA in the epididymis of mice.

Fetal and neonatal exposure to diethylstilbestrol (DES) is known to cause many abnormalities, such as cancer, in the male and female reproductive tracts later in life, and epigenetic mechanisms, such as DNA methylation, may be involved in these processes. In the present study, newborn C57BL/6 male mice were exposed to 3 mug of DES from postnatal days 1 to 5. Subsequently, the expression levels of the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b and the transcription factors Sp1 and Sp3, which have been reported to regulate the expression of Dnmts, were examined at days 5, 14 and 30. Furthermore, restriction landmark genomic scanning (RLGS), which can analyze genome-wide DNA methylation, was performed to clarify whether or not aberrant DNA methylation was present in the epididymis of the DES-treated mice at day 30. Increased expression of Dnmt3b was observed at days 5 and 14, followed by increased expression of Dnmt1 and Dnmt3a at day 30, as evaluated by real-time RT-PCR. The expression of Sp1 was also increased at day 30. The RLGS analysis revealed that 7 loci of the genomic DNA were demethylated and 1 locus was methylated in the epididymis of the DES-treated mice. Four of these loci specifically demethylated in DES-treated mice were cloned, and all were found to be located within CpG islands near genes. In conclusion, our results indicated the possibility that DES-induced abnormalities of reproductive organs are associated with altered expression levels of DNA methyltransferases and DNA methylation.

Estrogenic compounds inhibit gap junctional intercellular communication in mouse Leydig TM3 cells.

Some estrogenic compounds are reported to cause testicular disorders in humans and/or experimental animals by direct action on Leydig cells. In carcinogenesis and normal development, gap junctional intercellular communication (GJIC) plays an essential role in maintaining homeostasis. In this study, we examine the effects of diethylstilbestrol (DES, a synthetic estrogen), 17beta-estradiol (E(2), a natural estrogen), and genistein (GEN, a phytoestrogen) on GJIC between mouse Leydig TM3 cells using Lucifer yellow microinjection. The three compounds tested produced GJIC inhibition in the TM3 cells after 24 h. Gradually, 10 microM DES began to inhibit GJIC for 24 h and this effect was observed until 72 h. On the other hand, both 20 microM E(2) and 25 microM GEN rapidly inhibited GJIC in 6 h and 2 h, respectively. The effects continued until 24 h, but weakened by 72 h. Furthermore, a combined effect at microM level between DES and E(2) on GJIC inhibition was observed, but not between GEN and E(2). DES and E(2) showed GJIC inhibition at low dose levels (nearly physiological estrogen levels) after 72 h, but GEN did not. DES-induced GJIC inhibition at 10 pM and 10 microM was completely counteracted by ICI 182,780 (ICl), an estrogen receptor antagonist. On the other hand, the inhibitory effects on GJIC with E(2) (10 pM and 20 microM) and GEN (25 microM) were partially blocked by ICI or calphostin C, a protein kinase C (PKC) inhibitor, and were completely blocked by the combination of ICI and calphostin C. These results demonstrate that DES inhibits GJIC between Leydig cells via the estrogen receptor (ER), and that E(2) and GEN inhibit GJIC via ER and PKC. These estrogenic compounds may have different individual non-genotoxic mechanism including PKC pathway on testicular carcinogenesis or development.