Hideki Fukata

Necessity to Measure PCBs and Organochlorine Pesticide Concentrations in Human Umbilical Cords for Fetal Exposure Assessment

Three types of tissue samples—umbilical cord (UC), umbilical cord serum (CS), and maternal serum (MS)—have often been used to assess fetal exposure to chemicals. In order to know the relationship of contamination between mothers and fetuses, we measured persistent chemicals in comparable sets of the three tissue samples. Also, we analyzed the association between the chemicals in maternal and fetal tissues to know which tissue is the best sample for fetal exposure assessment. On a wet basis, the chemical concentrations were of the order MS > CS > UC, except for some chemicals such as cis-chlordane and endosulfan. On a lipid basis, the concentrations in UC were nearly equal or often higher than in MS, but the concentrations in CS were usually lower than in others. Hexachlorocyclohexanes and penta-, hexa-, and heptachlorinated biphenyls showed an association between the concentrations in UC versus MS, and UC versus CS. These chemicals also showed high correlation coefficients between the chemical concentrations in UC of first babies and maternal age. These chemicals were closely related to each other when grouped on the basis of their concentrations using cluster analysis. In conclusion, we insist that UC is the best sample to assess fetal contamination status of persistent chemicals. There is a possibility that the assessment based on the contamination levels in CS result in an underestimation.

Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences.

SummaryType II DNA topoisomerase has been isolated from inflorescences of cauliflower (Brassica oleracea var. botrytis) through a sequence of polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex, hydroxyapatite and phosphocellulose. The molecular weight of the native enzyme, based on sedimentation coefficient (9S) and gel filtration analysis (Stokes radius, 60 Å), was estimated to be 223 000. This enzyme was able to catalyze fully the relaxation of supercoiled DNA by breaking and then rejoining the double-stranded DNA. The breaking reaction was reversible by a change in salt concentrations. When an antitumor drug, 4′-(9-acridinylamino)-methanesulfon-m-anisidide, was added to the topoisomerase reaction, DNA cleavage fragments were accumulated; and this suggested that the drug interfered with the reaction at the rejoining step. This enzyme also catalyzed the formation of DNA catenanes in the presence of 8% polyethylene glycol or histone H1, while few catenanes were formed in the presence of spermidine, which was highly effective on a bacterial enzyme.

Identification and Characterization of Novel and Unknown Mouse Epididymis-Specific Genes by Complementary DNA Microarray Technology

Abstract 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, designatedSerpina1f(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 theSerpina1fmRNA concentration; this recovery was also observed with T alone. However, the recovery ofCuzd1andWfdc16mRNA concentrations was inadequate. Neonatal diethylstilbestrol treatment suppressed theCuzd1,Wfdc16, andSerpina1fmRNA 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.

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.

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.

Epigenetic alteration by the chemical substances, food and environmental factors

Epigenetic alteration is one of the most important mechanisms for gene regulation; however, it is not changes in gene function with DNA sequence changes. Recently, epigenetics were studied in the wide ranging fields of research. In the present review, we introduce recent studies on epigenetic alteration, especially DNA methylation, by chemical exposure, food intake and environmental factors. In addition, we introduced our results on alteration of DNA methylation by transient exposure of neonatal mice to diethylstilbestrol. As these data suggest that chemical exposure, food intake and environmental factors are responsible for epigenetic alteration, we insist the necessity of the new risk assessment focusing on epigenetic alteration.

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.

Assembly of nucleosome-like structures mediated by cauliflower DNA topoisomerase.

Nucleosome-like structures have been efficiently assembled in vitro by interaction of cauliflower histones, pBR322 DNA and cauliflower DNA topoisomerase, as assayed by supercoiling of relaxed circular DNA and by digestion with micrococcal nuclease. The optimum ionic strength for supercoiling was 150 mM KCl and the optimum weight ratio of histone to DNA was approximately 1.0. Four histones, H2A, H2B, H3 and H4, were necessary for the optimum assembling conditions, and the nucleosomes assembled protected DNA fragments of approximately 150 bp in length. It was found that cauliflower DNA topoisomerase acts not only as a DNA-relaxing enzyme but also as a chaperon factor for nucleosome assembly.

Template-specific inhibitor for DNA polymerase isolated from cauliflower inflorescence

A novel inhibitory factor which greatly inhibits DNA polymerase activity was isolated from the apical portion of the cauliflower inflorescence during purification of DNA polymerases. It can be adsorbed on a DEAE-cellulose column, but not on CM-Sephadex or DNA-cellulose. The factor exclusively inhibits the incorporation of [3H]dTTP into DNA when poly(rA, dT10) is used as the template primer, but not when activated DNA, heat-denatured DNA or native DNA is used as a template. The concentration of the factor in the reaction medium required for 50% inhibition is approx. 8 microgram/ml. The factor is heat-stable, is inactivated by trypsin, and has a maximum ultraviolet absorption at 278 nm. The molecular weight was estimated as 2500-3000 by Sephadex gel chromatography.