Tetrabromobisphenol A disrupts vertebrate development via thyroid hormone signaling pathway in a developmental stage-dependent manner.

Data concerning effects of tetrabromobisphenol A (TBBPA) on thyroid hormone (TH)-dependent vertebrate development have been limited, although TBBPA has been demonstrated in vitro to disrupt the TH signaling pathway at the transcriptional level. In this study, we investigated the effects of TBBPA on T3-induced and spontaneous Xenopus laevis metamorphosis, which share many similarities with TH-dependent development in higher vertebrates. In a 6-day T3-induced metamorphosis assay using premetamorphic tadpoles, 10-1000 nM TBBPA exhibited inhibitory effects on T3-induced expression of TH-response genes and morphological changes in a concentration-dependent manner, with a weak stimulatory action on tadpole development and TH-response gene expression in the absence of T3 induction. In a spontaneous metamorphosis assay, we further found that TBBPA promoted tadpole development from stage 51 to 56 (pre- and prometamorphic stages) but inhibited metamorphic development from stage 57 to 66 (metamorphic climax). These results strongly show that TBBPA, even at low concentrations, disrupts TH-dependent development in a developmental stage-dependent manner, i.e., TBBPA exhibits an antagonistic activity at the developmental stages when animals have high endogenous TH levels, whereas it acts as an agonist at the developmental stages when animals have low endogenous TH levels. Our study highlights the adverse influences of TBBPA on TH-dependent development in vertebrates.

Molecular characterization and mRNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones.

Like Xenopus laevis, some species of the Rana genus are also used to study endocrine disrupting chemicals (EDCs). Although ribosomal protein L8 (rpl8) is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rana, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rpl8 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rpl8 cDNA consisted of 919bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rpl8 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rpl8 gene was found in all tissues examined (brain, liver, intestine, tail, testis and ovary) during R. nigromaculata development. Finally, we investigated rpl8 expression under exposure to hormones. No change in rpl8 mRNA expression was found under exposure to thyroid hormone (T4) and estrogen (estradiol), whereas expression of the corresponding biomarker genes was induced. Our results show that rpl8 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rpl8 as a reference gene in other Rana species due to the high conservation of rpl8 among the Rana genus.

Molecular characterization and developmental expression patterns of thyroid hormone receptors (TRs) and their responsiveness to TR agonist and antagonist in Rana nigromaculata.

Considering some advantages of Rana nigromaculata as an experimental species, we propose that this species, like Xenopus laevis, could be used to assay thyroid hormone (TH) signaling disrupting actions. To validate the utilizability of R. nigromaculata, we investigated the responsiveness of R. nigromaculata to a TH receptor (TR) agonist (T3) and antagonist (amiodarone) by analyzing expression, based on characterizing TR cDNA and developmental expression patterns. With high levels of identity with the corresponding genes in X. laevis, both TRα and TRß in R. nigromaculata exhibited roughly similar developmental expression patterns to those of X. laevis, in spite of some species-specific differences. Both TRα and TRß expression had greater changes in the liver and intestine than in the tail and brain during metamorphosis. T3 exposure for 2days induced more dramatic increases of TRß expression in stage 27 than in stage 34 tadpoles but not in stage 42 tadpoles, showing that the responsiveness of R. nigromaculata to TH decreased with development and disappeared at the onset of metamorphic climax. Corresponding to greater changes of TRß expression in the liver and intestine than in the tail and brain during metamorphosis, the liver and intestine had higher responsiveness to exogenous T3 than the tail and brain. Amiodarone inhibited T3-induced TRß expression. Our results show that R. nigromaculata can be used as a model species for assaying TH signaling disrupting actions by analyzing TRß expression, and intestine tissues at stage 27 are ideal test materials due to high responsiveness and easy accessibility.

Effects of perfluorooctanesulfonate and perfluorobutanesulfonate on the growth and sexual development of Xenopus laevis.

Perfluorobutanesulfonate (PFBS), as a substitute for perfluorooctanesulfonate (PFOS), is widespread in the environment and biotic samples as well as PFOS. To investigate effects of PFOS and PFBS on the growth and sexual development of amphibians, we exposed Xenopus laevis tadpoles at a series of concentrations of PFOS and PFBS (0.1; 1; 100; 1,000 µg/l) as well as 17-beta-estradiol (E2, 100 ng/l) and 5 alpha-androstan-17-beta-ol-3-one (DHT, 100 ng/l) from stage 46/47 to 2 months postmetamorphosis. We found that neither PFOS nor PFBS had a significant effect on the survival and growth. However, they caused hepatohistological impairment at higher concentrations (100; 1,000 µg/l). Unlike E2, PFOS at all concentrations did not alter the sex ratio and induce intersex, but caused degeneration of spermatogonia in testes except for the lowest concentration. PFBS had no effect on the sex ratio and gonadal histology. PFOS and PFBS promoted expression of estrogen receptor (ER) and androgen receptor (AR), but not affected aromatase expression in the brain. The increase in expression of ER and AR suggests an increase in the responsiveness to the corresponding sex hormone and potential effects on sexual development. Our results show that PFBS as well as PFOS have adverse effects on hepato-histology and sexual development on X. laevis. Also, PFOS- and PFBS-induced increase in ER and AR expression highlights the need to further study effects of PFOS and PFBS on subsequently gonadal development, sexual dimorphism, and secondary sex characteristics in X. laevis. It is debatable that PFBS is widely used as a substitute of PFOS.

Multi-clinical index classifier combined with AI algorithm model to predict the prognosis of gallbladder cancer.

Objectives: It is significant to develop effective prognostic strategies and techniques for improving the survival rate of gallbladder carcinoma (GBC). We aim to develop the prediction model from multi-clinical indicators combined artificial intelligence (AI) algorithm for the prognosis of GBC. Methods: A total of 122 patients with GBC from January 2015 to December 2019 were collected in this study. Based on the analysis of correlation, relative risk, receiver operator characteristic curve, and importance by AI algorithm analysis between clinical factors and recurrence and survival, the two multi-index classifiers (MIC1 and MIC2) were obtained. The two classifiers combined eight AI algorithms to model the recurrence and survival. The two models with the highest area under the curve (AUC) were selected to test the performance of prognosis prediction in the testing dataset. Results: The MIC1 has ten indicators, and the MIC2 has nine indicators. The combination of the MIC1 classifier and the "avNNet" model can predict recurrence with an AUC of 0.944. The MIC2 classifier and "glmet" model combination can predict survival with an AUC of 0.882. The Kaplan-Meier analysis shows that MIC1 and MIC2 indicators can effectively predict the median survival of DFS and OS, and there is no statistically significant difference in the prediction results of the indicators (MIC1: χ2 = 6.849, P = 0.653; MIC2: χ2 = 9.14, P = 0.519). Conclusions: The MIC1 and MIC2 combined with avNNet and mda models have high sensitivity and specificity in predicting the prognosis of GBC.

Multi-Biomarkers Panel in Identifying Benign and Malignant Lung Diseases and Pathological Types of Lung Cancer.

With the discovery of many tumor markers, there are new strategies for the early diagnosis and treatment of lung cancer and the prediction of prognosis. We examined the multi-protein markers panel (4MP, consisting of Pro-SFTPB, CA125, Cyfra21-1, and CEA) diagnosis performance in differentiating benign and malignant lung diseases and identifying pathological types of lung cancer. Meantime, the complementary performance of three conventional tumor markers (NSE, SCC, and Pro-GRP) for 4MP was assessed. A total of 294 patients with lung cancer or benign lung disease are contained in this study. The AUCs of 4MP and 7MP (NSE, SCC, Pro-GRP, and 4MP) in distinguishing benign lung disease and lung cancer were 0.808 and 0.832, respectively. In distinguishing SQCLC and SCLC, the AUCs were 0.716 and 0.985, respectively. In distinguishing LADC and SCLC, the AUCs were 0.849 and 0.998, respectively. This study demonstrated that 4MP can distinguish lung cancer from benign disease. Traditional biomarkers NSE, SCC, and Pro-GRP can significantly improve the performance of 4MP in the differentiation of LADC, SQCLC, and SCLC, which is expected to contribute to the accurate diagnosis and personalized treatment of patients.

A New Method for the Detection of Colorectal Cancer and the Precancerous Lesions: Occult Blood Testing Combination with Promoter Methylation in the Fecal Sample.

Background: Noninvasive stool-based DNA methylation testing emerges as a new approach for detecting colorectal cancer (CRC). However, its feasibility for early detection of CRC and precancerous lesions in the Chinese population remains inconclusive. Methods: In this study, we establish a possibilities screening method (sDNA-FOBT) for detecting CRC and precancerous lesions (hyperplastic polyps [HP] and adenomas [AD]) and evaluate its detection performance in the Chinese population. This method combined a molecular assay of DNA methylation markers (BMP3, NDRG4, and SDC2) with the human hemoglobin test (FOBT) in stool samples. Results: The sensitivity of sDNA-FOBT was 85.42% for CRC, 85.71% for AD, and 28.21% for HP, respectively, at the specificity of 92%. The diagnostic efficacy of sDNA-FOBT for detecting CRC and precancerous lesions was significantly higher than FOBT alone (sensitivity: 61.70% vs. 51.06%, P<0.01; AUC: 0.78 vs. 0.72, P<0.001), especially for CRC (AUC: 0.91 vs. 0.86, P<0.001) and AD (AUC: 0.91 vs. 0.75, P<0.05). No significant difference was observed between the detection sensitivity of sDNA-FOBT and the clinical variables. Notably, compared with FOBT, sDNA-FOBT was more effective in the detection of CRC and precancerous lesions in the patients aged >50 y (62.34% vs 54.55%, P<0.05). Conclusion: Our results demonstrate that sDNA-FOBT is a promising method for screening CRC and precancerous lesions in the Chinese population. Further studies are required to validate the results in a larger sample capacity.

Auxiliary diagnosis of Lung Cancer on the basis of a Serum Protein Biomarker Panel.

Objectives: In this study, we established a serum protein biomarker panel (consisting of Pro-SFTPB, CA125, Cyfra21-1, and CEA) and evaluated the feasibility and performance for the auxiliary diagnosis of lung cancer in the Chinese population. Materials and Methods: The current study was a single-center study based on the Chinese population and performed in two cohorts (training cohort and validation cohort). Serum concentrations of Pro-SFTPB, CA125, Cyfra21-1, and CEA were measured by a bead-based flow fluorescence immunoassay. The discrimination performance of the model was assessed using sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve (AUC). Results: For the biomarker panel model, the AUC was 0.88 (95% CI, 0.85-0.91) in the training cohort and 0.90 (95% CI, 0.86-0.92) in the validation data cohort, which was significantly greater than the AUC of each biomarker alone. For the nodule risk model, the AUC was improved to 0.96 (95% CI, 0.94-0.98) in the training cohort and 0.95 (95% CI, 0.93-0.97) in the validation cohort. In addition, the biomarker panel model yielded an AUC of 0.78 (95% CI, 0.74-0.81) for stage I & II lung cancer, better than the performance of individual biomarker alone. Conclusions: It was demonstrated that 4-protein biomarker panel had a significant performance in identifying lung cancer patients from healthy controls, especially combining with the nodule size. Specifically, it yielded excellent discrimination for identifying early-stage lung cancer patients than individual biomarker alone. A future large-scale study is underway to further define the clinical application of this method for the early diagnosis of lung cancer among Chinese populations.

Accumulation of polybrominated diphenyl ethers in the brain compared with the levels in other tissues among different vertebrates from an e-waste recycling site.

This study aimed to investigate the accumulation of polybrominated diphenyl ethers (PBDEs) in the brain compared with that in other tissues among different vertebrates. We collected mice, chickens, ducks, frogs, and fish from an e-waste recycling region in Taizhou, China, and measured PBDE concentrations in brain, liver and muscle tissues. The levels of PBDE in the tissues of mice, chickens, ducks, frogs and fish ranged 0.45-206, 0.06-18.8, 1.83-112, 2.75-108, and 0.02-32.0 ng/g wet weight, respectively. Preferential distribution in the liver and muscle relative to the brain was observed for PBDEs in mice, chickens, ducks and frogs. However, a high retention in the brain compared to the liver and muscle was observed in fish. Comparison of the brain/liver concentration (B/L) ratios revealed differences in PBDEs accumulation in the brain among these vertebrates. PBDEs accumulation in the brain was greatest in fish, followed by frogs, while the lowest accumulation occurred in the brains of mammals and birds. The findings apparently coincided with the evolution of the blood-brain barrier (BBB) across vertebrates, i.e. the BBB of fish might be less efficient than those of mammals, birds and amphibian. Low brominated congeners (such as BDE-28, BDE-47 and BDE-99) were predominant in the brains of investigated vertebrates, whereas BDE-209 was most abundant in liver and muscle tissues of mice, chickens and ducks. Significant differences in B/L ratios among PBDE congeners were found in both mice and chickens (p < 0.05). Particularly in mice, the B/L ratios of PBDE congeners presented a declining trend with increased bromine number. Our findings suggested that low brominated congeners might have a higher capacity to penetrate the BBB and accumulate in the brain, whereas high brominated congeners such as BDE-209 might have less potency to pass through the barrier. Further experimental studies are needed to confirm our findings.

Low concentrations of dihydrotestosterone induce female-to-male sex reversal in the frog Pelophylax nigromaculatus.

Previous studies have demonstrated that some amphibian species can be sex-reversed by high concentrations of androgens. Little attention has focused on the effects of androgenic endocrine-disrupting chemicals (EDCs) on amphibians. The present study aimed to investigate the effects of lower concentrations of the androgenic EDC 5α-dihydrotestosterone (DHT) on gonadal differentiation and development in Pelophylax nigromaculatus, a true frog distributed widely in East Asia. Tadpoles at Gosner stage 24/25 were exposed to nominal concentrations of 40 ng/L, 400 ng/L, and 4000 ng/L DHT to complete metamorphosis. In all DHT treatment groups, males and ambiguous sexes were identified based on gonadal morphology, whereas no females were found; thus, all treatment groups exhibited male-skewed ratios compared with the control group. Gonadal histological examination revealed that ambiguous sexes displayed overall testicular structure with certain ovarian characteristics, demonstrating that DHT-induced sex-ambiguous gonads were incomplete ovary-to-testis reversals (IOTTRs). The expression levels of some ovary-biased genes in the IOTTRs were significantly higher than in the control testes but lower than in the control ovaries. These results show that low concentrations of DHT induced complete or incomplete female-to-male sex reversal in P. nigromaculatus, and incomplete sex reversal retained certain ovarian characteristics not only at gonadal morphological and histological levels but also at the molecular level. They present study highlights potential risks of DHT and other androgenic EDCs for P. nigromaculatus.

Low concentrations of 17ß-trenbolone induce female-to-male reversal and mortality in the frog Pelophylax nigromaculatus.

Trenbolone, as a growth promoter in animal agriculture, has become an environmental androgen in surface water. Here, we aimed to reveal the effects of 17ß-trenbolone on survival, growth, and gonadal differentiation in the frog Pelophylax nigromaculatus, which is widespread in East Asia and undergoing population decline. P. nigromaculatus tadpoles were exposed to 17ß-trenbolone (0.1, 1, 10 µg/L) from Gosner stage 24/25 to complete metamorphosis. We found that 17ß-trenbolone resulted in significantly high mortality in a concentration-dependent manner, with a decrease in body weight in the high concentration group compared with the solvent control. Based on gross gonadal morphology, no females were observed, instead of about 15% ambiguous sexes and 85% males, in all 17ß-trenbolone treatment groups. Like normal testes, the gonads with sex-ambiguous morphology exhibited testicular histology, showing that the sex-ambiguous gonads were incomplete ovary-to-testis reversals (IOTTRs) with certain ovarian morphological features. In the IOTTRs, the transcriptional levels of ovary-biased genes decreased drastically relative to normal ovaries, and even declined to the levels in normal testes. These observations confirmed that all test concentrations of 17ß-trenbolone resulted in 100% sex reversal, although some sex-reversed testes retained some ovarian characteristics at the morphological level. To our knowledge, this is the first report strongly demonstrating that trenbolone can cause female-to-male reversal in amphibians. Given that the lowest concentration tested is environmentally relevant, our study highlights the risks of trenbolone and other environmental androgens for P. nigromaculatus and other amphibians, in particular the species with high sensitivity of gonadal differentiation to androgenic chemicals.

Environmental (anti-)androgenic chemicals affect germinal vesicle breakdown (GVBD) of Xenopus laevis oocytes in vitro.

Progesterone-induced germinal vesicle breakdown (GVBD) of Xenopus oocytes in vitro was used to study endocrine disrupting activity of chemicals in previous studies. In this study, we investigated for the first time effects of environmental androgens on oocyte maturation and effects of anti-androgens on androgen-induced oocyte maturation, using Xenopus GVBD in vitro. Trenbolone and nandrolone, two environmental androgens, were found to induce Xenopus GVBD at low concentrations. The potential of trenbolone to induce GVBD was approximately 100-fold lower than that of testosterone, while nandrolone had a several-fold lower potential than testosterone. Our findings have aroused new concerns for effects of environmental androgens on amphibian oocyte maturation at environmentally relevant concentrations, and suggested that Xenopus GVBD can be used to test androgenic activity of suspicious environmental androgens. Androgen receptor (AR) antagonist flutamide at 10 µM only exhibited a weakly inhibitory effect on androgen-induced GVBD, while another known AR antagonist vinclozolin had no effect even at high concentrations. The results show that Xenopus GVBD is not sensitive to AR-mediated environmental anti-androgens. In contrast to flutamide and vinclozolin, methoxychlor (a weaker AR antagonist) inhibited dramatically androgen-induced GVBD, suggesting that androgen-induced Xenopus GVBD can be used to study non-AR-mediated effects of chemicals on oocyte maturation.