Shigeru Miyazaki

Perfluorinated organic compounds in human blood serum and seminal plasma: a study of urban and rural tea worker populations in Sri Lanka

Concentrations and accumulation of 13 fluorinated organic compounds (FOCs) in human sera and seminal plasma were measured in an Asian developing country, Sri Lanka. Six of the FOCs, PFOS (perfluorooctanesulfonate), PFHS (perfluorohexanesulfonate), PFUnA (perfluoroundecanoic acid), PFDA (perfluorodecanoic acid), PFNA (perfluorononanoic acid) and PFOA (perfluorooctanoic acid), were detected in all of the sera samples. Measurable quantities of two main perfluorosulfonates, PFOS and PFHS, were found in all seminal plasma samples. The detection frequency of the predominant perfluoroalkylcarboxylate, PFOA, in seminal plasma was >70%. Accumulation of PFOS in sera was significantly positively correlated with PFOA, PFHS and PFNA. Positive linear regressions were also found between PFNA and PFUnA and PFNA and PFDA suggesting that these compounds may have a similar origin of exposure and accumulation. Significantly positive associations were observed for partitioning of both PFOS and PFNA between sera and seminal plasma. The accumulation of FOCs was not significantly different in sera from Colombo (urban population) and Talawakele (rural conventional tea workers). However, the Haldummulla population (rural organic tea workers) had relatively lower exposure to FOCs compared to the other two groups, urban and rural conventional tea workers. Concentrations of FOCs in Sri Lanka were similar to those reported for industrialized countries suggesting that human exposure to such chemicals is widespread even in developing countries. The novel finding of FOCs in human seminal plasma implies that further studies are needed to determine whether long-term exposure in humans can result in reproductive impairments.

Differential expression of chicken hepatic genes responsive to PFOA and PFOS

The effects of PFOS and PFOA on the gene expression patterns of chickens that were exposed to either PFOS or PFOA at low doses were investigated with the use of microarray techniques. Twelve Genechip Chicken Genome Arrays were used to study hepatic gene expression in 6-week-old chickens (Gallus gallus) that were exposed to either PFOA (0.1, 0.5, or 5mg/mL), PFOS (0.02 or 0.1mg/mL), or a saline vehicle control (0.9% NaCl in Milli-Q water) via subcutaneous implantation of a 2mL osmotic pump for 4 weeks or for 4 weeks with a further 4 weeks of depuration. Over 240 and 480 genes were significantly affected by PFOS after 4 weeks of exposure and after 4 weeks of exposure with a further 4 weeks of depuration, respectively and over 290 and 320 genes were significantly affected by PFOA, correspondingly. For PFOS, the genes that were affected after 4 weeks of exposure were mainly related to the transport of electrons and oxygen, and the metabolism of lipids and fatty acids; while the genes that were affected after 4 weeks of exposure with a further 4 weeks of depuration were mainly related to the transport of electrons and ions, and protein amino acid phosphorylation and proteolysis. For PFOA, the genes that were affected after 4 weeks of exposure were related to the transport of ions, lipids, and electrons and cytochromes; while the genes that were affected after 4 weeks of exposure with a further 4 weeks of depuration were related to protein amino acid phosphorylation and proteolysis, the transport of ions, and the metabolism of fatty acids and lipids. The results also showed that the gene expression patterns between chickens that were treated with PFOS and those that were treated with PFOA were different, which points to the importance of the separate evaluation of the toxicities of PFOS and PFOA. Specifically, the gene expressions of CYP8B and NOV were studied.

Species-specific concentrations of perfluoroalkyl contaminants in farm and pet animals in Japan

The persistent metabolites of perfluorinated compounds (PFCs) which have been detected in the tissues of both humans and wildlife, and human contamination by PFCs suggest differences in the exposure patterns to these compounds. However, studies focused on identifying human exposure pathways to PFCs are scarce. To provide a preliminary assessment of PFCs in farm animals such as chicken, cattle, pigs, goats and horses, blood and liver samples were collected from various regions in Japan. Additionally, dog sera samples representing pet animals were also employed for analysis. Perfluorooctane sulfonate (PFOS) was the most prominent contaminant found in farm and pet animals, with mean sera PFOS concentrations (in decreasing order) of: chicken (5.8 ng/ml)>cattle (3.0 ng/ml)>goat (2.4 ng/ml)>horse (0.71 ng/ml)>pig (0.37 ng/ml). Chicken livers (67 ng/g) contained the highest mean PFOS concentration among the farm animals, followed by those of pigs (54 ng/g) and cattle (34 ng/g). In comparison to PFOS levels in farm animals, the detected levels of other PFCs were not significant. The high levels of PFOS found in cattle fetal livers suggest that PFOS crosses the placental barrier to enter fetal circulation. The consumption of chicken by humans might produce higher PFOS exposure in humans compared to that in farm animals; however, the current levels of PFOS in farm animals in Japan were lower than those reported in fish and wild animals. Elevated concentrations of both PFOS (25 ng/ml) and perfluorohexane sulfonate (PFHxS; 10 ng/ml) were found in dog sera, indicating that further studies are needed to identify PFC sources in the human environment.

Depuration kinetics and tissue disposition of PFOA and PFOS in white leghorn chickens (Gallus gallus) administered by subcutaneous implantation.

Elimination kinetics and tissue disposition of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in male chickens (Gallus gallus) was determined following exposure by subcutaneous implantation. Chickens were exposed to two levels of PFOA or PFOS for 4wk and then allowed to depurate for an additional 4wk. These exposures did not cause any statistically significant changes in body index, clinical biochemistry or histology among treatments relative to the controls (p>0.05), except that concentrations of total cholesterol and phospholipids were less in chickens exposed to PFOS. The elimination rate constant for PFOA (0.150+/-0.010d(-1)) was approximately six-fold greater than that of PFOS (0.023+/-0.004d(-1)). The greatest concentrations of PFOA and PFOS were found in kidney and liver, respectively. The organ to blood ratio of PFOS concentration was increased after the whole experiment, indicating the importance of organ partitioning of PFOS in elimination kinetics. The depuration half-life of PFOA (t(1/2)=4.6d) and PFOS (t(1/2)=125d) in chickens was calculated.

Fate of maize intrinsic and recombinant genes in calves fed genetically modified maize Bt11.

The presence of maize intrinsic and recombinant cry1Ab genes in the gastrointestinal (GI) contents, peripheral blood mononuclear cells (PBMC), and visceral organs of calves fed genetically modified Bt11 maize was examined by PCR in a subchronic 90-day performance study. Samples were collected from six Japanese Black/Holstein calves fed Bt11 maize and from six calves fed non-Bt maize. Fragments of maize zein (Ze1), invertase, chloroplast, and cry1Ab were detected inconsistently in the rumen fluid and rectal contents 5 and 18 h after feeding. The chloroplast DNA fragments of ribulose-1,5-bisphosphate carboxylase/oxygenase and tRNA were detected inconsistently in the PBMC, the visceral organs, and the longissimus muscle, while the cry1Ab gene was never detected in PBMC or in the visceral organs. These results suggest that feed-derived maize DNA was mostly degraded in the GI tract but that fragmented DNA was detectable in the GI contents as a possible source of transfer to calf tissues. These results also suggest that the recombinant cry1Ab genes were not transferred to the PBMC and tissues of calves fed Bt11 maize.

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets.

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against single-stranded DNA and cleaved caspase-3. The number of TUNEL-positive cells in the thymus and Peyers patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor (TNF)-α in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1β mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1β and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-α decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.

Lolitrem B residue in fat tissues of cattle consuming endophyte-infected perennial ryegrass straw.

Lolitrems are neurotoxins found in endophyte-infected perennial ryegrass. Lolitrems, primarily lolitrem B, are the causative agents of ryegrass staggers in livestock. To guarantee the safety of meat produced from cattle consuming endophyte-infected perennial ryegrass, lolitrem B concentrations in tissues of Japanese Black cattle were determined using high-performance liquid chromatography. Lolitrem B was not detected in muscle, liver, kidney, or cerebrum of a Japanese Black cow with signs of ryegrass staggers. In contrast, perirenal fat contained 210 ppb lolitrem B. Three cows that received half as much perennial ryegrass straw as the cow with ryegrass staggers showed no clinical signs of ryegrass staggers. However, low concentrations of lolitrem B (less than 150 ppb) were detected in their fat tissue. These observations indicate that human exposure to the neurotoxic effect of lolitrem B through beef is unlikely. The amount of lolitrem B consumed by cattle can be estimated by the determination of lolitrem B in fat tissue.

Differential induction of cytochrome P450 1A1 and 1B1 mRNA in primary cultured bovine hepatocytes treated with TCDD, PBDD/Fs and feed ingredients.

This study investigates the dose-dependent expression of CYP1A1 and CYP1B1 in primary cultured bovine hepatocytes exposed to TCDD, several polybrominated dibenzo-p-dioxins and furans (PBDD/Fs) congeners and fish oil used as animal feed ingredients to identify their dioxin-like potentials. Hepatocytes were isolated from calf liver, cultured and treated for 24h with the target compounds or extracts. Quantitative real time polymerase chain reaction analysis (qRT-PCR) showed that relative mRNA levels for CYP1A1 and CYP1B1 exhibited a dose-dependent induction by TCDD. The EC(50) of the TCDD concentration for CYP1A1 expression was approximately 4-fold less than that of CYP1B1. The estimated dioxin-like toxic potential of PBDD/Fs could be ranked in the following order: 2,3,7,8-TBDD>1,2,3,7,8-PBDF>2,3,4,7,8-PBDF>1,2,3,6,7,8-HBDD. A good correlation was also observed in HRGC/HRMS-derived TEQs in fish oil samples and relative CYP1A1 mRNA induction in bovine hepatocytes treated with purified fish oil extracts. The data suggested that quantification of biomarker regulations in primary cultured hepatocytes could represent an effective tool for both the screening and study of various chemical entities in larger animals.

Accumulation of polychlorinated naphthalenes in domestic animal related samples

Concentrations of polychlorinated naphthalene (PCN) congeners were measured in domestic animal related samples such as feed ingredients, mixed feed and animal fat. Mean concentrations of total PCNs in feed ingredients ranged from 500 to 1500 pg g(-1) lipid wt with a high concentration found in fish meal. Total PCN concentrations were similar among mixed feeds, which ranged from 98 to 110 pg g(-1) lipid wt. The total PCN concentration in chickens was more than twice the amount in pigs. Tetra-CNs were the predominant homologues in all samples. Biomagnification of higher chlorinated PCN congeners, especially penta- and hexa-CNs, was a few fold greater in chickens compared to pigs. The estimated concentrations of 2,3,7,8-tetrachloro dibenzo-p-dioxin equivalents (TEQs) of some selected PCNs in feed ingredients, mixed feeds, chickens and pigs were 0.008 to 0.063, 0.001 to 0.002, 0.033 and 0.011 pg g(-1) lipid wt, respectively. Based on predicted luciferase inducing potency for each PCN congener, the estimated PCN-TEQs in feed ingredients and animal fat were similar to those that were estimated from selected PCNs.

Simple Capillary Electrophoretic Determination of Soluble Oxalate and Nitrate in Forage Grasses

A simple capillary electrophoretic method is described for the simultaneous determination of soluble oxalate and nitrate in forage grasses. Grass samples were ground and extracted with water. The extracts were filtered and submitted to capillary electrophoresis. Electrophoresis was performed in a 75 μm ×50 cm fused silica capillary with 30 mM sodium sulfate containing an electroosmotic flow modifier under constant voltage at —8 kV Separated oxalate and nitrate were detected with direct UV absorption at 214 nm. The present method can be used for routine monitoring of the concentration of soluble oxalate and nitrate in grasses.