Acute and chronic toxicity tests of systemic insecticides, four neonicotinoids and fipronil, using the tadpoles of the western clawed frog Silurana tropicalis.

Extensive use of neonicotinoids and fipronil, which are popular systemic insecticides used in Japanese rice paddies, has raised concerns about their impacts on nontarget aquatic organisms such as amphibians. This study employed premetamorphic tadpoles of Silurana tropicalis and addressed the toxicity of four neonicotinoids (acetamiprid, clothianidin, dinotefuran, and imidacloprid) and fipronil. Acute toxicity tests were conducted under a 96-h semistatic exposure regime and median lethal concentration (LC50) values were calculated at 24-h intervals. All LC50 values of the four neonicotinoids exceeded 100 mg/L, suggesting their low acute toxicity to amphibians. Fipronil yielded much lower LC50 values (3.00-1.34 mg/L) and was highly toxic compared to the four neonicotinoids. Additionally, exposure to fipronil at >1 mg/L induced axial malformations, suggesting its teratogenicity. However, the LC50 values of fipronil were three orders of magnitude higher than the realistic concentrations in paddy water. Chronic toxicity tests were conducted with morphometric, gravimetric, and thyroid-histological endpoints. Premetamorphic tadpoles were exposed to each insecticide at two test concentrations: 0.1 and 1.0 mg/L for the four neonicotinoids; and 1/100 and 1/10 of the 96-h LC50 value for fipronil. Exposure to each insecticide continued until all tadpoles in the control reached late prometamorphic stages or the initial stage of metamorphic climax. At test termination, all insecticides showed no significant differences in any of the endpoints between the respective controls and chemical exposure groups. Overall, our results suggest that these insecticides alone do not directly affect amphibians through their larval stages at concentrations that likely occur in paddy water.

Chronic toxicity of 1,3,5-triazine herbicides in the postembryonic development of the western clawed frog Silurana tropicalis.

Seven 1,3,5- triazine (s-triazine) herbicides (ametryn, prometryn, dimethametryn, simazine, atrazine, propazine, and cyanazine) were tested using an amphibian (Silurana tropicalis) metamorphosis assay focusing on morphometric, gravimetric, and thyroid-histological endpoints. Premetamorphic tadpoles were exposed to each s-triazine at 2 concentrations between 1/1000 and 1/10 of the 96-h acute toxicity values, until all tadpoles in the control group reached either the late prometamorphosic stages or the initial stage of metamorphic climax. All s-triazines tested induced significant retardation in growth and development at the higher concentrations (0.2-1.0mg/L), and some of them induced similar effects even at the lower concentrations (0.02-0.1mg/L) while each showing a linear dose-response. Total size of the thyroid glands tended to be reduced corresponding to the delayed development, but without showing histomorphological lesions typical of anti-thyroid chemicals. These consistent results suggest that the s-triazines can act as a chemical stressor inhibiting tadpole growth and development, possibly without disrupting the thyroid axis. In addition, tadpoles exhibiting spinal curvatures appeared in either one or both of the lower and higher concentration groups for each s-triazine tested. The incidence rate in the s-triazine exposure groups where tadpoles with scoliosis were observed ranged from 3.3% to 63.3%, some of which were significantly higher than that in the respective control groups (0-6.7%). It is speculated that the s-triazines may promote to occur axial malformations in developing tadpoles.

Prediction of the preoperative chemoradiotherapy response for rectal cancer by peripheral blood lymphocyte subsets.

BACKGROUND: Although neoadjuvant chemoradiotherapy (CRT) has become a standard procedure to downstage locally advanced rectal cancer prior to surgery, markers to predict the response to CRT have not been fully identified. The aim of this study was to identify predictive factors of response to CRT, especially focusing on peripheral blood leukocyte subsets. METHODS: A total of 45 consecutive patients diagnosed with primary rectal cancer were prospectively enrolled and received CRT followed by curative resection. The numbers of each lymphocyte subset in peripheral blood pre- and post-CRT were analyzed using flow cytometry. According to the pathological response to CRT, patients were classified into high (Hi-R) and low (Lo-R) response groups. RESULTS: Hi-R cases had significantly higher numbers of pre-CRT lymphocytes (p = 0.018), T lymphocytes (p = 0.009) and helper T lymphocytes (Th lymphocytes, p = 0.015) compared to the Lo-R cases. With the receiver-operating characteristic curve for numbers of pre-CRT T lymphocytes, the area under the curve (AUC) was 0.733, and the optimal cutoff value was 1196/µl, with 76.5% sensitivity, 67.8% specificity, 59.1% positive and 82.6% negative predictive values. The numbers of pre-CRT Th lymphocytes and cytotoxic lymphocytes were both independent predictors of the high CRT response in the multivariate analysis. CONCLUSIONS: In addition to the direct cytotoxicity of CRT, recent studies have demonstrated the induction of an immunological host response, which also contributed to the tumor regression induced by CRT. Our result suggested the potential role of circulating T lymphocytes in predicting the response to CRT in colorectal cancer patients.

Temsirolimus and chloroquine cooperatively exhibit a potent antitumor effect against colorectal cancer cells.

PURPOSE: Temsirolimus (TEM) is a novel, water-soluble mammalian target of rapamycin (mTOR) inhibitor that has shown activity against a wide range of cancers in preclinical models, but its efficacy against colorectal cancer (CRC) has not been fully explored. METHODS: We evaluated the antitumor effect of TEM in CRC cell lines (CaR-1, HT-29, Colon26) in vitro and in vivo. In vitro, cell growth inhibition was assessed using a MTS assay. Apoptosis induction and cell cycle effects were measured using flow cytometry. Modulation of mTOR signaling was measured using immunoblotting. Antitumor activity as a single agent was evaluated in a mouse subcutaneous tumor model of CRC. The effects of adding chloroquine, an autophagy inhibitor, to TEM were evaluated in vitro and in vivo. RESULTS: In vitro, TEM was effective in inhibiting the growth of two CRC cell lines with highly activated AKT, possibly through the induction of G1 cell cycle arrest via a reduction in cyclin D1 expression, whereas TEM reduced HIF-1α and VEGF in all three cell lines. In a mouse subcutaneous tumor model, TEM inhibited the growth of tumors in all cell lines, not only through direct growth inhibition but also via an anti-angiogenic effect. We also explored the effects of adding chloroquine, an autophagy inhibitor, to TEM. Chloroquine significantly potentiated the antitumor activity of TEM in vitro and in vivo. Moreover, the combination therapy triggered enhanced apoptosis, which corresponded to an increased Bax/Bcl-2 ratio. CONCLUSIONS: Based on these data, we propose TEM with or without chloroquine as a new treatment option for CRC.

Tsunami: ocean dynamo generator.

Secondary magnetic fields are induced by the flow of electrically conducting seawater through the Earth's primary magnetic field ('ocean dynamo effect'), and hence it has long been speculated that tsunami flows should produce measurable magnetic field perturbations, although the signal-to-noise ratio would be small because of the influence of the solar magnetic fields. Here, we report on the detection of deep-seafloor electromagnetic perturbations of 10-micron-order induced by a tsunami, which propagated through a seafloor electromagnetometer array network. The observed data extracted tsunami characteristics, including the direction and velocity of propagation as well as sea-level change, first to verify the induction theory. Presently, offshore observation systems for the early forecasting of tsunami are based on the sea-level measurement by seafloor pressure gauges. In terms of tsunami forecasting accuracy, the integration of vectored electromagnetic measurements into existing scalar observation systems would represent a substantial improvement in the performance of tsunami early-warning systems.

Changes in the plasma levels of cytokines/chemokines for predicting the response to chemoradiation therapy in rectal cancer patients.

In the present study, we aimed to characterize the predictive value of cytokines/chemokines in rectal cancer (RC) patients receiving chemoradiation therapy (CRT). Blood samples were obtained pre- and post-CRT from 35 patients with advanced RC, who received neoadjuvant CRT followed by surgery, and the correlation between plasma levels of cytokines/chemokines and the response to CRT was analyzed. The pre-CRT levels of soluble CD40-ligand (sCD40L) and the post-CRT levels of chemokine ligand-5 (CCL-5) were significantly associated with the depth of tumor invasion and with venous invasion. In addition, a significant decrease in sCD40L and CCL-5, as well as in platelet counts, was associated with a favorable response to CRT. A significant correlation between pre-CRT platelet counts and sCD40L was observed in patients with a favorable response. By contrast, higher post-CRT interleukin (IL)-6 was associated with a poor response. Platelets, immune system and cancer cells, cross-linked through various cytokines/chemokines, appear to play an important role in the response to CRT, and by understanding their roles, new approaches for the improvement of the therapy might be proposed.

Application of an amphibian (Silurana tropicalis) metamorphosis assay to the testing of the chronic toxicity of three rice paddy herbicides: simetryn, mefenacet, and thiobencarb.

We examined the chronic toxicity of three rice paddy herbicides (simetryn, mefenacet, and thiobencarb) using an amphibian (Silurana tropicalis) metamorphosis assay (a 28-day semistatic test under an individual-separated exposure system). Each herbicide was tested at two concentrations (1/100 and 1/10 of the 96-h LC50 value reported previously) with morphometric, gravimetric, and thyroid-histological endpoints. Simetryn caused significant retardation in growth and development at both test concentrations (0.04 and 0.40mg/L), as indicated by significantly shorter total body lengths and hind limb lengths, smaller wet body masses, and delayed developmental stages compared to those observed in the control tadpoles. However, no clear histopathology was observed in the thyroid glands of the tadpoles exposed to simetryn. These results suggest that simetryn can act as a chemical stressor retarding tadpole growth and development without disrupting thyroid functions, even at 1/100 of the 96-h LC50 value. In addition, scoliosis near the tail base was observed in the tadpoles exposed to 0.40mg/L of simetryn at a significantly high incidence (7/30=23.3%). Therefore, simetryn can also act as a teratogen inducing axial malformations at 1/10 of the 96-h LC50 value. During the 28 days of exposure, neither mefenacet (0.03 and 0.30mg/L) nor thiobencarb (0.008 and 0.080mg/L) induced any abnormalities, although the test concentrations measured immediately before the solution renewals decreased to nearly 50 percent of the nominal concentrations since day 14. Because the concentrations tested for simetryn are likely to occur in paddy water, wild anuran tadpoles in paddy water may therefore be adversely impacted by simetryn.

Hypoxia enhances colon cancer migration and invasion through promotion of epithelial-mesenchymal transition.

BACKGROUND: A hypoxic environment exists in most solid tumors because in rapidly growing tumors, the development of angiogenic vasculature is heterogenous, usually not enough to overcome the necessary oxygen supply. In an ischemic condition, cancer cells develop escape mechanisms to survive and leave the unfavorable environment. That result in the acquisition of increased potential for local invasion and evasion to distant organs. However, the escape mechanisms of cancer cells from hypoxic stress have not been fully characterized. MATERIALS AND METHODS: The human colon cancer cell line LoVo was cultured in hypoxia, and the adhesive and migratory properties were analyzed. The expression of cell surface and cytoplasmic molecules was also investigated. RESULTS: Under hypoxic conditions, cells developed epithelial-mesenchymal transition. The expression levels of α2, α5, and ß1 integrins were significantly upregulated and, as a consequence, the ability to adhere to and migrate on collagen and fibronectin was increased. On the other hand, the expression of 67-kDa laminin receptor and the abilities to adhere to and migrate on laminin were decreased. Additionally, the expression of CXCR4 was significantly increased on cells cultured in hypoxia, and the chemotactic activity to stromal cell-derived factor 1α was remarkably increased. CONCLUSIONS: Hypoxic stress induced active epithelial-mesenchymal transition in colon cancer cells, with the typical morphologic and functional changes. These morphologic and functional changes of ß1 integrins, the 67-kDa laminin receptor, and CXCR4 may be essential for the acquisition of the invasive and metastatic features in colorectal cancer.

Examination of an amphibian metamorphosis assay under an individual-separated exposure system using Silurana tropicalis tadpoles.

We examined the validity of an amphibian (Silurana tropicalis) metamorphosis assay (a 28-day semistatic test) under an individual-separated exposure system, where tadpoles were individually held in small glass beakers. We first conducted a comparative rearing experiment for 28 days between this exposure system and the traditional individual-grouped exposure system, both of which held 30 tadpoles (stages 49 and 50) in dechlorinated tap water (a control solution). The former system served to reduce interindividual variability in regard to three morphological measures (developmental stage, hind limb length, and total body length). Under this system, we tested thyroxine (T4, 1µg/L) and propylthiouracil (PTU, 75mg/L) for 28 days of exposure. The morphological data collected at 7-day intervals indicated that significant metamorphic acceleration and retardation were consistently induced in the tadpoles exposed to T4 and PTU, respectively. In addition, the thyroid glands of the tadpoles exposed to T4 and PTU clearly exhibited atrophy and hypertrophy accompanied with severe follicular cell hyperplasia, respectively. Our results are in agreement with the historical data generated from previous studies employing the traditional exposure system, thus indicating the validity of our alternative testing protocol.

Orexin 2 receptor as a potential target for immunotoxin and antibody-drug conjugate cancer therapy.

Targeting tumor-specific receptors is a promising approach for cytotoxic agents. The orexin 2 receptor (OX2R) has reportedly been expressed in a few types of cancer, but not in normal, cells. This study aimed to explore and assess the expression levels of OX2R in a wide range of cancer cell lines and clinical samples to identify its localization. To analyze OX2R expression, we developed a polyclonal antibody specific to OX2R by immunizing two rabbits with a peptide cocktail. A total of 36 cancer cell lines were employed for reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, and 221 samples from various tissue arrays were used for the immunohistochemistry of OX2R expression. OX2R was identified in three cancerous cell lines, from the gallbladder, squamous cell carcinoma of the head and neck (SCCHN) and glioblastoma. With clinical samples of tissue arrays, 69/221 (31.2%) samples reacted positively with the OX2R antibody. We confirmed its presence on the cell membrane. In conclusion, OX2R was identified on several cancer cells as well as clinical samples. Further studies with larger numbers of clinical samples are required to confirm the statistical significance of the presence and relationships of OX2R with tumor histology. Results of the current study suggested that OX2R is a potent target for immunotoxin or antibody-drug conjugate (ADC) cancer therapy on OX2R-positive cancer cells.

Resistance of colon cancer to 5-fluorouracil may be overcome by combination with chloroquine, an in vivo study.

Autophagy is a complex of adaptive cellular response that enhances cancer cell survival in the face of cellular stresses such as chemotherapy. Recently, chloroquine diphosphate (CQ), a widely used antimalarial drug, has been studied as a potential inhibitor of autophagy. Here, we aimed to investigate the role of CQ in potentiating the effect of 5-fluorouracil (5-FU), the chemotherapeutic agent of first choice for the treatment of colorectal cancer, in an animal model of colon cancer. The mouse colon cancer cell line colon26 was used. For the in-vivo study, colon26 cells were injected subcutaneously into BALB/c mice, which were treated with saline as a control, CQ (50 mg/kg/day), 5-FU (30 mg/kg/day), or the combination therapy (CQ plus 5-FU). The tumor volume ratio and body weight were monitored. After the sacrifice, tumor tissue protein extracts and tumor sections were prepared and subjected to immunoblotting for the analysis of autophagy-related and apoptosis-related proteins, and the terminal transferase uridyl end labeling assay. The combination of CQ resulted in the inhibition of 5-FU-induced autophagy and a significant enhancement in the 5-FU-induced inhibition of tumor growth. Furthermore, the combination treatment of CQ and 5-FU resulted in a significant increase in the ratio of apoptotic cells compared with other treatments. The expression levels of the proapoptotic proteins, namely Bad and Bax, were increased by the CQ treatment in the protein extracts from tumors. Our findings suggest that the combination therapy of CQ and 5-FU should be considered as an effective strategy for the treatment of colorectal cancer.

Elevated neutrophil to lymphocyte ratio predicts poor prognosis in advanced colorectal cancer patients receiving oxaliplatin-based chemotherapy.

BACKGROUND: The aim of this study was to assess whether the neutrophil to lymphocyte ratio (NLR) and other laboratory markers may predict the prognosis of advanced colorectal cancer (CRC) patients receiving palliative chemotherapy. METHODS: The study population included 50 patients with far advanced or recurrent unresectable CRC who received oxaliplatin-based combination chemotherapy as first-line treatment in our hospital between June 2005 and November 2010. Seven clinical variables and 7 laboratory indices before chemotherapy were evaluated retrospectively as the possible prognostic factors of overall and progression-free survival. RESULTS: During the study period, 27 patients (54%) died of CRC. Elevated NLR (≥4.0) was observed in 15 patients (30%). By univariate analysis, elevated NLR, performance status and hypoalbuminemia were significantly associated with both poor overall and progression-free survivals. Multivariate analysis showed that elevated NLR (hazard ratio 4.39, 95% confidence interval 1.82-10.7; p = 0.0013) and thrombocytosis (hazard ratio 5.02, 95% confidence interval 1.69-13.4; p = 0.0066) were independently associated with overall survival. CONCLUSION: Elevated NLR is a powerful predictor of poor response to oxaliplatin-based chemotherapy in patients with unresectable CRC. The ratio is a simply accessible and inexpensive but useful biomarker in CRC patients receiving chemotherapy.

SN-38 overcomes chemoresistance of colorectal cancer cells induced by hypoxia, through HIF1alpha.

BACKGROUND: Cancer cells can acquire resistance to therapy under hypoxic condition. We aimed to investigate the mechanisms regulating chemoresistance induced by hypoxia. MATERIALS AND METHODS: Human colorectal cancer cells, HT-29 and SW480, were cultured under hypoxic conditions and the sensitivity to 5-fluorouracil (FU), oxaliplatin, and SN-38 (active metabolite of irinotecan) was tested. The cell cycle was evaluated by flow cytometry after staining of cells with propidium iodide (PI). hypoxia-inducible factor 1α (HIF-1α) expression was evaluated by western blot analysis. RESULTS: Hypoxia induced strong G(0)/G(1) cell cycle arrest of cancer cells and abrogated the cytotoxic effects of 5-FU and oxaliplatin, but not that of SN-38. This effect was dependent on the significant inhibition of the accumulation of HIF-1α in cancer cells cultured under hypoxia by SN-38. Neither 5-FU nor oxaliplatin affected HIF-1α expression. CONCLUSION: SN-38, through inhibition of HIF-1α can overcome chemoresistance under hypoxic conditions of colon cancer cells.

The current status of umbilical cord blood collection in Japanese medical centers: survey of obstetricians.

As the first step of UCB banking, UCB collection has an important role in banking procedures. The aim of this study was to reveal the current status of UCB collection and discuss the management of the UCB bank. We conducted a questionnaire survey at medical centers collecting UCB, followed by semi-structured interviews with some respondents. Out of 38 institutes, 11 respondents (28.9%) thought that collection of UCB in addition to their routine medical services puts a burden on physicians. The obstetricians involved in the UCB collection are generally willing to participate in the procedure under current circumstances at medical institutes.

A single replacement of histidine to arginine in EGFR-lytic hybrid peptide demonstrates the improved anticancer activity.

We previously reported that novel targeted "hybrid peptide" in which epidermal growth factor receptor (EGFR) binding peptide was conjugated with lytic-type peptide had selective cytotoxic activity to EGFR expressing cancer cell lines, and in vivo analysis revealed that this EGFR-lytic peptide displayed significant antitumor activity in a xenograft model of human breast cancer which was resistant to tyrosine kinase inhibitor drugs. As an attempt to improve the selective anticancer activity of EGFR-lytic peptide, we modified the EGFR-binding peptide through introducing the mutation of amino acid according to biophysical analysis by biomolecular interaction and circular dichroism (CD) spectra. When cytotoxic activity of EGFR-lytic or EGFR(2R)-lytic hybrid peptides was investigated in various human cancer and normal cell lines, it was demonstrated that EGFR(2R)-lytic, in which second histidine (H) of EGFR-binding peptide was replaced to arginine (R) had 1.2-1.9-fold higher cytotoxic activity than that of original EGFR-lytic peptide. In vivo analysis also revealed that this modified peptide displayed significant antitumor activity at as low as 1 mg/kg dosage. These results suggest that mutated arginine on EGFR-lytic peptide produces higher binding ability to EGFR on cancer cells, and thereby the improved anticancer activity.

Radiosensitization of human breast cancer cells to ultraviolet light by 5-fluorouracil.

Ultraviolet light B (UVB) phototherapy is widely used to treat dermatological diseases and therefore may be a potential optional strategy in the treatment of a skin lesion infiltrated by a malignant tumor. Currently, little is known regarding the effect of UVB phototherapy on human breast cancer cells. The present study aimed to investigate the effect of UVB phototherapy, as well as the potential effect of 5-fluorouracil (5-FU), the first-line anticancer drug for breast cancer, on radiosensitizing MCF-7 human breast cancer cells, in an attempt to develop new therapeutic strategies for the treatment of locoregional recurrence of breast cancer. MCF-7 cells were incubated in the presence of 5-FU for 48 h, and UVB irradiation at 750 mJ/cm(2) was administered in the midterm of 5-FU treatment. The viability of MCF-7 cells was analyzed by the trypan blue staining method. Apoptosis was quantified by flow cytometry and Hoechst 33258 staining. The cell cycle was evaluated by flow cytometry after the staining of cells with propidium iodide. The combination treatment of 5-FU and UVB resulted in a strong potentiation of the inhibitory effect of MCF-7 cell growth, dependent on the intra-S phase cell cycle arrest and induction of apoptosis, when compared to treatment with 5-FU or UVB alone. In conclusion, 5-FU sensitized human breast cancer cells to UVB phototherapy, and this combination therapy is an effective and promising strategy for the treatment of breast cancer, particularly for locoregional recurrence.

Vitellogenin, a biomarker for environmental estrogenic pollution, of Reeves' pond turtles: analysis of similarity for its amino acid sequence and cognate mRNA expression after exposure to estrogen.

Vitellogenin (VTG), a biomarker for environmental estrogenic pollution, can be detected in the bloodstream of oviparous animals before morphological and functional abnormalities appear due to exposure to environmental estrogens. Reports observing VTG in turtles have been limited. We therefore cloned and sequenced a partial cDNA of VTG in Reeves' pond turtle, Chinemys reevesii. The cloned cDNA fragment possessed the start codon and 2,229 bp, encoding 743 amino acid residues. A sequence of deduced amino acid from the cDNA did not contain a high serine content, such as that which exists in phosvitin. Two N-glycosylation sites were found in the sequence. The sequence was compared to those of two birds (chicken and herring gull), one amphibian (Xenopus), and five fishes (carp, zebrafish, eel, haddock, and red seabream). The C. reevesii VTG was similar to that of herring gull (78%, value of positives), chicken (76%), Xenopus (69%), eel (63%), red seabream (62%), haddock (62%), carp (62%), and zebrafish (61%). The phylogenetic tree showed that C. reevesii VTG existed between the amphibian and birds, and it was present far from fish VTGs. A reverse transcription-polymerase chain reaction method was employed to detect the mRNA expression of the C. reevesii VTG through the use of primers designed from our sequence. The VTG mRNA expression (292 bp) was proven in the total RNA extraction from the liver of the juvenile turtles which were treated with estradiol-17beta. The information herein would be useful for ecotoxicological studies using freshwater turtles and these findings are expected to contribute positively towards wildlife conservation.

Cross-reactivity of a polyclonal antibody against Chinemys reevesii vitellogenin with the vitellogenins of other turtle species: Chelydra serpentina , Macrochelys temminckii , and Pelodiscus sinensis.

Vitellogenin (VTG), a yolk-precursor protein in oviparous vertebrates, is a useful biomarker for reproductive physiology and environmental estrogenic pollution. To examine interspecific applicability of an enzyme-linked immunosorbent assay (ELISA) for quantifying Chinemys reevesii VTG, we observed cross-reactivity between a polyclonal antibody against Chinemys reevesii VTG and the VTGs from other turtle species: Chelydra serpentina (Chelydridae), Macrochelys temminckii (Chelydridae), and Pelodiscus sinensis (Trionychidae), which are phylogenetically distant from Chinemys reevesii (Geoemydidae). The VTGs of the three species were induced by injecting estradiol 17beta into the turtles and purified by using the EDTA-MgCl(2) precipitation method. The purified VTG appeared as a 200-kDa protein in sodium dodecylsulfate polyacrylamide gel electrophoresis, indicating that the molecular mass of the VTGs of the three species was similar to that of Chinemys reevesii VTG. The purified VTGs were serially diluted (0.004-2 mug/ml) and applied to the ELISA. Although the VTGs of the two chelydrid turtles showed cross-reactivity in a concentration-dependent manner, the degree of cross-reactivity was only 22.8-41.2% (mean=30.0%) and 19.7-53.0% (mean=33.2%) for Chelydra serpentina VTG and Macrochelys temminckii VTG, respectively. The ELISA may therefore be theoretically applicable to measure relative levels of the VTGs of these two species, but the absolute concentration values may be inaccurate. Pelodiscus sinensis VTG showed almost no cross-reactivity (8.0-9.7%, mean=8.9%) at any concentration tested, thus indicating the inapplicability of the ELISA to quantify Pelodiscus sinensis VTG. There are thus limitations in extending the applicability of the ELISA across species, even within the order Testudines.

A field study on serum vitellogenin levels in male Reeves' pond turtles (Chinemys reevesii) from estrogen-contaminated sites and a reference site.

To ascertain whether wild male turtles were influenced by environmental estrogens, we examined serum vitellogenin (VTG) levels of male Reeves' pond turtles (Chinemys reevesii) collected from four study sites (A-D) in Kyoto, Japan. Sites A-C, which were impacted by domestic or industrial wastewater and effluents from sewage treatment plants, were chosen as contaminated sites, and site D was intended as a reference site. This contaminated/reference site characterization was confirmed by measuring estrogenic activities of the water samples collected at each site for over a year. Serum VTG levels in the turtles were quantified by an enzyme-linked immunosorbent assay established previously. Estrogenic activities of the water samples were measured using a previously validated yeast two-hybrid assay and expressed as the estradiol-17beta equivalent. Estrogenic activity was observed at high levels at sites A-C, but was almost undetected at site D throughout the sampling period: the mean and range were 0.74 (<0.07-2.1), 0.52 (0.17-1.6), 1.7 (<0.07-7.3), and 0.07 (<0.07-0.62) ng/l at sites A-D, respectively. Significant differences were found only in site D versus sites A, B, and C. Therefore, site D and sites A-C were confirmed to be a reference site and contaminated ones, respectively. Overall, 320 male turtles were captured and examined. The majority of the turtles showed normal VTG values (0.10-0.74 microg/ml). Although only five turtles from sites A-C showed unusually high VTG values (1.1-5.9 microg/ml, nearly one order of magnitude higher than normal values but much lower than values in adult females), there was no significant difference in the incidence of these high values between sites A-C and site D. Moreover, among the five turtles, one turtle was captured again 2 months later, but its VTG value dropped to the normal level. The unusually high VTG values may therefore be transient elevation caused by incidental and/or individually specific agents. Excluding the unusually high values, the mean serum VTG (accompanied with the range) was 0.22 (0.10-0.74), 0.27 (0.11-0.62), 0.27 (0.17-0.68), and 0.23 (0.10-0.57) microg/ml at sites A-D, respectively. There were no significant differences in the mean VTG values among sites A-D. Although it should be clarified how and why the unusually high VTG values occurred at sites A-C, our results suggested that wild male C. reevesii turtles would not be significantly affected by xenobiotic estrogens at environmentally relevant levels in terms of serum VTG elevation.