Variation in the sensitivity of intestine and skin of Bufo gargarizans and Rana chensinensis tadpoles in relation to zinc exposure.

Zinc (Zn) contaminants in the aquatic environment have an intricate impact on amphibians. Amphibian gut and skin microbiota are participated in regulating their normal physiological functions. Here, we investigated the effects of Zn on the gut and skin tissues and microbiota of Bufo gargarizans and Rana chensinensis tadpoles using histological methods and 16S rRNA sequencing technology. Our results showed a decrease in the height of enterocytes and skin epithelial cells after Zn treatment. Furthermore, Zn exposure elicited alterations in the composition and structure of the gut and skin microbiota at the phylum and genus levels in Bufo gargarizans and Rana chensinensis tadpoles. The feature predictions revealed an elevation in the abundance of potentially pathogenic bacteria and stress-tolerant bacteria in the gut and skin of both tadpoles after zinc exposure. We also speculated that microbiota from various species and organs exhibit varying degrees of sensitivity to zinc based on the functional predictions results. In the context of increasing environmental pollution and the global amphibians decline, our research enriches the current understanding of effects of zinc on amphibian microbiota and provides new framework for artificial breeding and amphibian conservation.

Gut microbiota-bile acid crosstalk contributes to intestinal damage after nitrate exposure in Bufo gargarizans tadpoles.

Bile acids (BAs) are amphipathic steroid acids whose production and diversity depend on both host and microbial metabolism. Nitrate (NO3-) is a widespread pollutant in aquatic ecosystems, which can cause rapid changes in microbial community structure and function. However, the effect of gut microbiota reshaped by nitrate­nitrogen (NO3-N) on BAs profiles remains unclarified. To test this, intestinal targeted BAs metabolomics and fecal metagenomic sequencing were performed on Bufo gargarizans tadpoles treated with different concentrations of NO3-N. NO3-N exposure induced a reduction in the abundance of microbiota with bile acid-inducible enzymes (BAIs) and/or hydroxysteroid dehydrogenases (HSDHs), thus inhibiting the conversion of primary BAs to secondary BAs. Inhibition of BAs biotransformation decreased protective hydrophilic BAs (UDCA) and increased toxic hydrophobic BAs (CA and CDCA), which may contribute to intestinal histopathological damage. Moreover, we found that NO3-N treatment increased microbial virulence factors and decreased Glycoside hydrolases, further highlighting the deleterious risk of NO3-N. Overall, this study shed light on the complex interactions of NO3-N, gut microbiota, and BAs, and emphasized the hazardous effects of NO3-N pollution on the health of amphibians.

Response characteristic and potential molecular mechanism of tail resorption in Bufo gargarizans after exposure to lead and copper, alone or combined.

Tail resorption during amphibian metamorphosis is one of the most dramatic processes that is obligatorily dependent on thyroid hormone (TH). Heavy metals could result in thyroid gland damages and disturb TH homeostasis. Lead (Pb) and copper (Cu) often co-exist in natural aquatic ecosystems. However, there is still little information on how tail resorption responds to alone or combined exposure to Pb and Cu. Our study investigated the effects of Pb and Cu alone or combined exposure on the morphological parameters of the tail, histological changes of thyroid gland and tail, and gene expression programs involved in cell death of the tail in Bufo gargarizans tadpoles at the climax of metamorphosis. Results demonstrated that Pb, Cu and Pb-Cu mixture exposure resulted in a significantly longer tail compared with control. Damages to notochord, muscle, skin and spinal cord of the tail were found in Pb and Cu exposure groups. The colloid area, the height of follicular cells and number of phagocytic vesicles of thyroid gland in Pb-Cu mixture exposure groups were significantly reduced. In addition, the expression levels of TH, apoptosis, autophagy, degradation of cellular components and oxidative stress-related genes in the tail were significantly altered following Pb and Cu exposure. The present work revealed the relationship between environmental pollutants and tail resorption, providing scientific basis for amphibian protection.

[Preparation of Chitosan-modified Biochar and Its Adsorption Mechanism for Cd2+ in Aqueous Solution].

Chitosan-modified biochar （CBC） was prepared as a low-cost and highly efficient adsorbent for Cd2+ in aqueous solutions. Batch adsorption experiments were conducted to evaluate the adsorption performance. Characterization experiments with SEM-EDS， FTIR， and XPS were used to analyze the surface microstructure and chemical composition of the adsorbent. The results showed that the adsorption performance of CBC was remarkably improved by the introduction of surface functional groups （-OH， -C=O， and -NH2）. The pseudo-second-order kinetic model and Langmuir model were better for describing the kinetics and isotherms for Cd2+ adsorption onto CBC， indicating that the adsorption rate was determined by the active sites and controlled by monolayer chemisorption. The adsorption process was endothermic spontaneous， and the key mechanisms involved complexation， precipitation， cation exchange， and cation-π bonds. After five instances of adsorption-desorption cycles， the adsorption capacity of CBC for Cd2+ still remained above 80% of the initial adsorption capacity， indicating that CBC had a favorable recyclability. The current work embodies the concept of green chemistry， and the prepared chitosan-modified biochar was a promising adsorbent for the removal of Cd2+ in wastewater and soil.

FAM76B regulates NF-κB-mediated inflammatory pathway by influencing the translocation of hnRNPA2B1.

FAM76B has been reported to be a nuclear speckle-localized protein with unknown function. In this study, FAM76B was first demonstrated to inhibit the NF-κB-mediated inflammatory pathway by affecting the translocation of hnRNPA2B1 in vitro. We further showed that FAM76B suppressed inflammation in vivo using a traumatic brain injury (TBI) mouse model. Lastly, FAM76B was shown to interact with hnRNPA2B1 in human tissues taken from patients with acute, organizing, and chronic TBI, and with different neurodegenerative diseases. The results suggested that FAM76B mediated neuroinflammation via influencing the translocation of hnRNPA2B1 in vivo during TBI repair and neurodegenerative diseases. In summary, we for the first time demonstrated the role of FAM76B in regulating inflammation and further showed that FAM76B could regulate the NF-κB-mediated inflammatory pathway by affecting hnRNPA2B1 translocation, which provides new information for studying the mechanism of inflammation regulation.

Impairment of bile acid metabolism and altered composition by lead and copper in Bufo gargarizans tadpoles.

Lead (Pb) and copper (Cu) are two common heavy metal contaminants in environments, and liver is recognized as one of the main target organs for toxicity of Pb and Cu in animal organisms. Bile acids play a critical role in regulating hepatic metabolic homeostasis by activating farnesoid X receptor (Fxr). However, there were few studies on the interactions between bile acids and liver pathology caused by heavy metals. In this work, the histopathological changes, targeted metabolome and transcriptome responses in the liver of Bufo gargarizans tadpoles to Pb and/or Cu were examined. We found that exposure to Pb and/or Cu altered the hepatic bile acid profile, resulting in increased hydrophobicity and toxicity of the bile acid pool. And the expression of genes involved in bile acid metabolism and their downstream signaling pathways in the liver were significantly altered by Pb and/or Cu exposure. The alteration of bile acid profiles and the expression of genes related to bile acid metabolism might induce oxidative stress and inflammation, ultimately inducing hepatocyte injury observed in the histological sections. To our knowledge, this is the first study to provide histological, biochemical, and molecular evidence for establishing the link between Pb and Cu exposure, disturbances in hepatic bile acid metabolism, and liver injury.

Intestinal response of Rana chensinensis larvae exposed to Cr and Pb, alone and in combination.

Although numerous investigations on the adverse impact of Cr and Pb have been performed, studies on intestinal homeostasis in amphibians are limited. Here, single and combined effects of Cr (104 µg/L) and Pb (50 µg/L) on morphological and histological features, bacterial community, digestive enzymes activities, as well as transcriptomic profile of intestines in Rana chensinensis tadpoles were assessed. Significant decrease in the relative intestine length (intestine length/snout-to-vent length, IL/SVL) was observed after exposure to Pb and Cr/Pb mixture. Intestinal histology and digestive enzymes activities were altered in metal treatment groups. In addition, treatment groups showed significantly increased bacterial richness and diversity. Tadpoles in treatment groups were observed to have differential gut bacterial composition from controls, especially for the abundance of phylum Proteobacteria, Firmicutes, Verrucomicrobia, Actinobacteria, and Fusobacteria as well as genus Citrobacter, Anaerotruncus, Akkermansia, and Alpinimonas. Moreover, transcriptomic analysis showed that the transcript expression profiles of GPx and SOD isoforms responded differently to Cr and/or Pb exposure. Besides, transcriptional activation of pro-apoptotic and glycolysis-related genes, such as Bax, Apaf 1, Caspase 3, PK, PGK, TPI, and GPI were detected in all treatment groups but downregulation of Bcl2 in Pb and Cr/Pb mixture groups. Collectively, these results suggested that Cr and Pb exposure at environmental relevant concentration, alone and in combination, could disrupt intestinal homeostasis of R. chensinensis tadpoles.

Copper-mediated shifts in transcriptomic responses of intestines in Bufo gargarizans tadpoles to lead stress.

The differential transcriptomic responses of intestines in Bufo gargarizans tadpoles to Pb alone or in the presence of Cu were evaluated. Tadpoles were exposed to 30 µg/L Pb individually and in combination with Cu at 16 or 64 µg/L from Gosner stage (Gs) 26 to Gs 38. After de novo assembly, 105,107 unigenes were generated. Compared to the control group, 7387, 6937, and 11139 differentially expressed genes (DEGs) were identified in the treatment of Pb + Cu0, Pb + Cu16, and Pb + Cu64, respectively. In addition, functional annotation and enrichment analysis of DEGs revealed substantial transcriptional reprogramming of diverse molecular and biological pathways were induced in all heavy metal treatments. The relative expression levels of genes associated with intestinal epithelial barrier and bile acids (BAs) metabolism, such as mucin2, claudin5, ZO-1, Asbt, and Ost-ß, were validated by qPCR. This study demonstrated that Pb exposure induced transcriptional responses in tadpoles, and the responses could be modulated by Cu.

Effects of temperature on metamorphosis and endochondral ossification in Rana chensinensis tadpoles.

Temperature is one of the important factors affecting the growth, development, and metamorphosis of amphibians. Endochondral ossification during metamorphosis plays a crucial role in amphibian survival and adaptation on land. In this study, we explored the effects of different temperature treatments on the growth, development, and endochondral ossification of Rana chensinensis tadpoles during metamorphosis. The results showed that high temperature exposure may affect the skeletal development of tadpoles during metamorphosis, such as reduction of bone length and ossification of limbs, thyroid gland damage and change of ossification-related genes expression levels，and ultimately affect the movement and survival of tadpoles in the terrestrial environment. These results provide an experimental reference for further research on the effects of temperature on amphibian growth and development and provide an important theoretical basis for the decline of the amphibian population caused by temperature.

Lead and copper led to the dysregulation of bile acid homeostasis by impairing intestinal absorption in Bufo gargarizans larvae: An integrated metabolomics and transcriptomics approach.

Bile acids, as metabolic regulators and signaling molecules, play key roles in the regulation of host metabolism and immune responses. Heavy metals such as lead (Pb) and copper (Cu) are widespread environmental pollutants that threaten public health. However, the effects of heavy metals on bile acid metabolism and the underlying molecular mechanisms remain unclear, particularly for ecologically important amphibian species. In the present research, the effects of exposure to environmentally-relevant concentrations of Pb (250 µg/L), Cu (50 µg/L), and a mixture of both (Mix) on bile acid metabolism and the underlying molecular mechanisms in the intestines of Bufo gargarizans larvae were comprehensively investigated using histopathology, metabolomics and transcriptomics analysis. Our results suggested that Pb and/or Cu caused histopathological damage to the intestine and liver, such as decreased intestinal epithelial cell height and dilated hepatic sinusoid. The total bile acid level was decreased in the Pb and Mix exposure groups but elevated in the Cu treatment. A significant decrease in the ratio of conjugated to unconjugated bile acids was present in all treatment groups. Also, the level of GCA was increased while TCA and TCDCA were decreased in all exposure groups. In addition, exposure to Pb and Cu altered the expression levels of genes related to intestinal absorption. For example, mrp2, mrp3 and aqp4 had higher expression in the Pb and Mix treatment groups, and aqp1 and mrp4 were increased in the Cu treatment group. Overall, we speculated that the dysregulation of bile acid homeostasis induced by Pb and Cu exposure may be due to impaired intestinal absorption. These findings raise further concerns about the hazards of Pb and/or Cu in influencing bile acid metabolism that might lead to the development of metabolic diseases and inflammatory disorders.

Comparison of the characteristics of gut microbiota response to lead in Bufo gargarizans tadpole at different developmental stages.

In amphibians, lead (Pb) exposure could alter the composition and structure of gut microbiota, but changes involving microbiota of several successive phases following Pb exposure have been less studied. In the present study, we compared the effects of Pb exposure on morphological parameters and gut microbiota of Bufo gargarizans at Gosner stage (Gs) 33, Gs36, and Gs42. Our results showed that total length (TL), snout-vent length (SVL), and body wet weight (TW) of B. gargarizans at Gs33, as well as TL and SVL at Gs42, were significantly increased after Pb exposure. In addition, high-throughput sequencing analysis indicated that gut microbiota has distinct responses to Pb exposure at different developmental stages. The diversity of gut microbiota was significantly reduced under Pb exposure at Gs33, while it was significantly increased at Gs42. In terms of community composition, Spirochaetota, Armatimonadota, and Patescibacteria appeared in the control groups at Gs42, but not after Pb treatment. Furthermore, functional prediction indicated that the relative abundance of metabolism pathway was significantly decreased at Gs33 and Gs36, and significantly increased at Gs42. Our results fill an important knowledge gap and provide comparative information on the gut microbiota of tadpoles at different developmental stages following Pb exposure.

Effects of temperature on intestinal microbiota and lipid metabolism in Rana chensinensis tadpoles.

Climate change such as global warming is considered a major threat to amphibians. The guts of amphibians are home to trillions of microbes, which are key regulators of gastrointestinal digestion and play a crucial role in lipid metabolites. The aim of this study was to evaluate the effect of temperature change on intestinal microbiota and lipid metabolism in Rana chensinensis tadpoles. Morphological and intestinal microbiota data of R. chensinensis larvae exposed to different temperatures (15 °C, 21 °C, and 26 °C) were measured. The results show that the warm temperature causes histological damage to the intestinal epithelium. In addition, temperature treatments alter the diversity and composition of gut microbes in R. chensinensis tadpoles. At the phylum level of intestinal microbial community, Campilobacterota was detected only in the warm group. At the genera level, unclassified_f__Enterobacteriaceae was markedly declined in the warm group but was notably enriched in the cold group. For lipid metabolism-related genes, the expression levels of GPR109A, HDAC1, and APOA-I decreased significantly in both warm and cold treatment groups, while the expression levels of CLPS and LIPASE increased significantly. Collectively, these observations demonstrated that warm and cold temperatures may reduce the immune capacity of tadpoles by changing the composition of intestinal microorganisms and the expression of genes related to lipid metabolism, affecting the survival of tadpoles.

Lead and copper influenced bile acid metabolism by changing intestinal microbiota and activating farnesoid X receptor in Bufo gargarizans.

Lead (Pb) and copper (Cu) are ubiquitous metal contaminants and can pose a threat to ecosystem and human health. Bile acids have recently received considerable attention for their role in the maintenance of health. However, there were few studies on whether Pb and Cu affect bile acid metabolism in amphibians. In this study, a combination approach of histological analysis, targeted metabolomics, 16S rDNA sequencing and qPCR was used to explore the impacts of Pb, Cu and their mixture (Mix) on bile acid in Bufo gargarizans tadpoles. The results showed that Pb, Cu, and Mix resulted in intestinal damage and altered the bile acid profiles. Specifically, Pb and Mix exposure decreased total bile acid concentrations while increased toxic bile acid levels; in contrast, Cu exposure increased total bile acid levels. And hydrophilic bile acids were reduced in all treated tadpoles. Moreover, Pb and/or Cu changed the composition of intestinal microbiota, especially Clostridia, Bacteroides and Eubacterium involved in bile acid biotransformation. qPCR revealed that the decreased total bile acid concentrations in Pb- and Mix-treated tadpoles were most likely attributed to the activation of intestinal farnesoid X receptor (Fxr), which suppressed bile acid synthesis and reabsorption. While activated fxr in the Cu treatment group may be a regulatory mechanism in response to increased bile excretion, which is a detoxification route of tadpoles under Cu stress. Collectively, Pb, Cu and Mix changed bile acid profiles by affecting intestinal microbial composition and activating Fxr signaling. This study provided insight into the impacts of Pb and Cu on bile acid metabolism and contributed to the assessment of the potential ecotoxicity of heavy metals on amphibians.

Effects of waterborne Pb/Cu mixture on Chinese toad, Bufo gargarizans tadpoles: morphological, histological, and intestinal microbiota assessment.

Coexistence of heavy metals in aquatic environments exert complex effects on amphibians. Here, the adverse effects of Pb (0.14 µM) combined with Cu at concentrations of 0, 0.25, and 1.0 µM were investigated in Bufo gargarizans tadpoles. Tadpoles were chronically exposed from Gosner stage (Gs) 26 to Gs 38, and morphology of tadpoles as well as intestinal histology and bacterial community were assessed. Our results indicated that Pb+Cu1.0 exposure induced significant retardation of somatic mass, total length, intestine mass, and intestine length as well as intestinal histological alterations. Pb+Cu0.25 and Pb+Cu1.0 exposure were associated with the loss of gut bacterial diversity. Proteobacteria and Bacteroidetes were two dominant phyla in tadpoles independently of heavy metal exposure, but the abundance of Proteobacteria increased significantly in Pb+Cu1.0 group and Bacteroidetes decreased significantly in all treatment groups. Furthermore, functional prediction indicated that metabolic disorders were associated with Pb+Cu0.25 and Pb+Cu1.0 exposure. Overall, relative limited shifts in intestinal bacterial diversity, composition, and functionality caused by Pb+Cu0 exposure, while coexistence of Pb and Cu induced gut dysbiosis and might further cause disturbance of metabolic homeostasis. The findings of this study provide insights into the effects of Pb and Cu coexistence on the health of amphibians.

Effects of cadmium exposure on thyroid gland and endochondral ossification in Rana zhenhaiensis.

Discovery of elevated concentrations of cadmium in the natural environment has increased awareness because of their potential threats. Amphibians are negatively affected due to their moderate sensitivity to cadmium. Here, we conduct acute and subchronic toxicity tests to examine whether, and to what extent, cadmium exposure disturbs metamorphosis, growth, and kinetic ability of Rana zhenhaiensis. We set different concentration treatment groups for the subchronic toxicity test (0, 10, 40, 160 µg Cd L-1). Our findings demonstrate that cadmium exposure reduces growth parameters and the cumulative metamorphosis percent of R. zhenhaiensis. Decreases in follicular size and follicular epithelial cell thickness of thyroid gland are found in the treatment group. Further, subchronic exposure to cadmium decreases ossification ratio of hindlimbs in all treatment. Also, adverse effects of cadmium exposure on aquatic tadpoles can result in the reduced physical parameters and weak jumping ability in adult frogs. In this sense, our study suggests that cadmium adversely influences body condition and metamorphosis of R. zhenhaiensis, damages thyroid gland and impairs endochondral ossification. Meanwhile, we speculated that cadmium-damaged thyroid hormones inhibit skeletal development, resulting in the poor jumping ability, which probably leads to reduced survival of R. zhenhaiensis.

Using Physical Organic Chemistry Knowledge to Predict Unusual Metabolites of Synthetic Phenolic Antioxidants by Cytochrome P450.

Biotransformation, especially by human CYP450 enzymes, plays a crucial role in regulating the toxicity of organic compounds in organisms, but is poorly understood for most emerging pollutants, as their numerous "unusual" biotransformation reactions cannot retrieve examples from the textbooks. Therefore, in order to predict the unknown metabolites with altering toxicological profiles, there is a realistic need to develop efficient methods to reveal the "unusual" metabolic mechanism of emerging pollutants. Combining experimental work with computational predictions has been widely accepted as an effective approach in studying complex metabolic reactions; however, the full quantum chemical computations may not be easily accessible for most environmentalists. Alternatively, this work practiced using the concepts from physical organic chemistry for studying the interrelationships between structure and reactivity of organic molecules, to reveal the "unusual" metabolic mechanism of synthetic phenolic antioxidants catalyzed by CYP450, for which the simple pencil-and-paper and property-computation methods based on physical organic chemistry were performed. The phenol-coupling product of butylated hydroxyanisole (BHA) (based on spin aromatic delocalization) and ipso-addition quinol metabolite of butylated hydroxytoluene (BHT) (based on hyperconjugative effect) were predicted as two "unusual" metabolites, which were further confirmed by our in vitro analysis. We hope this easily handled approach will promote environmentalists to attach importance to physical organic chemistry, with an eye to being able to use the knowledge gained to efficiently predict the fates of substantial unknown synthesized organic compounds in the future.

Effects of nitrite exposure on metamorphosis and skeletal development of Bufo gargarizans.

Nitrite, as a part of nitrogen cycle, is one of the most common toxic compounds in aquatic ecosystems. Since skeletal development is an essential process during amphibian metamorphosis, exposure of larval amphibians to nitrite might disrupt skeletal development. To evaluate whether nitrite affects skeletal development of amphibian larvae, Bufo gargarizans larvae at Gs26 were exposed to 10, 100, 500 and 1000 µg/L nitrite-nitrogen (NO2-N) in the present study. The metamorphosis rate, body weight, body length, forelimb length and hindlimb length of B. gargarizans exposed to NO2-N were decreased. The microscopic structures of thyroid gland were altered under NO2-N exposure at Gs42. The skeletal lengths of the humerus, femur and fibulare of tadpole at Gs42 were significantly reduced under 100, 500 and 1000 µg/L NO2-N treatment groups, and the lengths of humerus, tibia-fibula and tibiale of tadpole at Gs46 were significantly reduced under 1000 µg/L NO2-N treatment groups. In addition, the expression levels of thyroid hormone (TH) and endochondral ossification-related genes of tadpoles at Gs42 and Gs46 were tested by qRT-PCR. Overall, NO2-N exposure could affect the expressions of these genes and then may influence the activity and function of thyroid gland, further disturbing the amphibian metamorphosis and skeletal development of amphibian larvae.

Computational Investigation of the Bisphenolic Drug Metabolism by Cytochrome P450: What Factors Favor Intramolecular Phenol Coupling.

Intramolecular phenol coupling reactions of alkaloids can lead to active metabolites catalyzed by the mammalian cytochrome P450 enzyme (P450); however, the mechanistic knowledge of such an "unusual" process is lacking. This work performs density functional theory computations to reveal the P450-mediated metabolic pathway leading from R-reticuline to the morphine precursor salutaridine by exploring possible intramolecular phenol coupling mechanisms involving diradical coupling, radical addition, and electron transfer. The computed results show that the outer-sphere electron transfer with a high barrier (>20.0 kcal/mol) is unlikely to happen. However, for inter-sphere intramolecular phenol coupling, it reveals that intramolecular phenol coupling of R-reticuline proceeds via the diradical mechanism consecutively by compound I and protonated compound II of P450 rather than the radical addition mechanism. The existence of a much higher radical rebound barrier than that of H-abstraction in the quartet high-spin state can endow the R-reticuline phenoxy radical with a sufficient lifetime to enable intramolecular phenol coupling, while the H-abstraction/radical rebound mode with a negligible rebound barrier leading to phenol hydroxylation can only happen in the doublet low-spin state. Therefore, the ratio [coupling]/[hydroxylation] can be approximately reflected by the relative yield of the high-spin and low-spin H-abstraction by P450, which thus can provide a theoretical ratio of 16:1 for R-reticuline, which is in accordance with previous experimental results. Especially, the high rebound barrier of the phenoxy radical derived from the weak electron-donating ability of the phenoxy radical is revealed as an intrinsic nature. Therefore, the revealed intramolecular phenol coupling mechanism can be potentially extended to several other bisphenolic drugs to infer groups of unexpected metabolites in organisms.

Effects of Separate and Combined Exposure of Cadmium and Lead on the Endochondral Ossification in Bufo gargarizans.

Cadmium (Cd) and lead (Pb) are ubiquitous in aquatic environments and most studies have examined the potential effects of Cd or Pb alone on aquatic organisms. In the present study, chronic effects of Cd and Pb, alone and in combination, on Bufo gargarizans were investigated by exposing embryos to these contaminants throughout metamorphosis. Significant reductions in body mass and snout-to-vent length were observed in B. gargarizans at Gosner stage 42 (Gs 42) and Gs 46 exposed to a Cd/Pb mixture. Single and combined exposure with Cd and Pb induced histological alterations of the thyroid gland characterized by reduced colloid area and thickness of epithelial cells. There was a significant decrease in the maximum jump distance of froglets exposed to Cd alone and the Cd/Pb mixture, and the jumping capacity showed a positive correlation with hind limb length and tibia/fibula. Moreover, single metals and their mixture induced reduction of endochondral bone formation in B. gargarizans. Transcriptomic and real-time quantitative polymerase chain reaction results showed that genes involved in skeletal ossification (TRα, TRß, Dio2, Dio3, MMP9, MMP13, Runx1, Runx2, and Runx3) were transcriptionally dysregulated by Cd and Pb exposure alone or in combination. Our results suggested that despite the low concentration tested, the Cd/Pb mixture induced more severe impacts on B. gargarizans. In addition, the Cd/Pb mixture might reduce chances of survival for B. gargarizans froglets by decreasing size at metamorphosis, impaired skeletal ossification, and reduction in jumping ability, which might result from dysregulation of genes involved in thyroid hormone action and endochondral ossification. The findings obtained could add a new dimension to understanding of the mechanisms underpinning skeletal ossification response to heavy metals in amphibians. Environ Toxicol Chem 2022;41:1228-1245. © 2022 SETAC.

Effects of perchlorate and exogenous T4 exposures on development, metamorphosis and endochondral ossification in Bufo gargarizans larvae.

Several endocrine-disrupting chemicals (EDCs) have been proven to interfere with the physiological function of thyroid hormone (TH), which affected growth and development. However, few studies have investigated the effects of EDCs on TH axis with consequence for skeletal development in amphibians. This study thus examined the potential role of perchlorate and T4 in growth, development and endochondral ossification during metamorphosis of Bufo gargarizans. Our studies showed that NaClO4 treatment caused weight gain and delayed the developmental stage in B. gargarizans tadpoles, while T4 decreased body size and survival rate, accelerated metamorphic duration and increased the risk of early death. Histological sections suggested that NaClO4 and T4 treatments caused damages to thyroid tissue, such as decreased thyroid gland size, follicle size, colloid area, the height of follicular epithelial cells and the number of follicles. In addition, the double skeletal staining and RT-qPCR showed that NaClO4 and T4 treatments inhibited the endochondral ossification by regulating TH synthesis (TRs, Dios) and endochondral ossification-related genes (MMPs, Runxs, VEGFs and VEGFRs) expression levels, which might affect terrestrial locomotion and terrestrial life. Altogether, these thyroid injury and gene expression changes as caused by NaClO4 and T4 may have an influence on development and endochondral ossification during the metamorphosis of amphibians.