Protocol for a comprehensive pipeline to study ancient human genomes.

Ancient genomics has revolutionized our understanding of human evolution and migration history in recent years. Here, we present a protocol to prepare samples for ancient genomics research. We describe steps for releasing DNA from human remains, DNA library construction, hybridization capture, quantification, and sequencing. We then detail procedures for mapping sequence reads and population genetics analysis. This protocol also outlines challenges in extracting ancient DNA samples and authenticating ancient DNA to uncover the genetic history and diversity of ancient populations. For complete details on the use and execution of this protocol, please refer to Tao et al.1.

Ancient genome of the Chinese Emperor Wu of Northern Zhou.

Emperor Wu (, Wudi) of the Xianbei-led Northern Zhou dynasty, named Yuwen Yong (, 543-578 CE), was a highly influential emperor who reformed the system of regional troops, pacified the Turks, and unified the northern part of the country. His genetic profile and physical characteristics, including his appearance and potential diseases, have garnered significant interest from the academic community and the public. In this study, we have successfully generated a 0.343×-coverage genome of Wudi with 1,011,419 single-nucleotide polymorphisms (SNPs) on the 1240k panel. By analyzing pigmentation-relevant SNPs and conducting cranial CT-based facial reconstruction, we have determined that Wudi possessed a typical East or Northeast Asian appearance. Furthermore, pathogenic SNPs suggest Wudi faced an increased susceptibility to certain diseases, such as stroke. Wudi shared the closest genetic relationship with ancient Khitan and Heishui Mohe samples and modern Daur and Mongolian populations but also showed additional affinity with Yellow River (YR) farmers. We estimated that Wudi derived 61% of his ancestry from ancient Northeast Asians (ANAs) and nearly one-third from YR farmer-related groups. This can likely be attributed to continuous intermarriage between Xianbei royal families, and local Han aristocrats.1,2 Furthermore, our study has revealed genetic diversities among available ancient Xianbei individuals from different regions, suggesting that the formation of the Xianbei was a dynamic process influenced by admixture with surrounding populations.

Genomic formation of Tibeto-Burman speaking populations in Guizhou, Southwest China.

Sino-Tibetan is the most prominent language family in East Asia. Previous genetic studies mainly focused on the Tibetan and Han Chinese populations. However, due to the sparse sampling, the genetic structure and admixture history of Tibeto-Burman-speaking populations in the low-altitude region of Southwest China still need to be clarified. We collected DNA from 157 individuals from four Tibeto-Burman-speaking groups from the Guizhou province in Southwest China. We genotyped the samples at about 700,000 genome-wide single nucleotide polymorphisms. Our results indicate that the genetic variation of the four Tibeto-Burman-speaking groups in Guizhou is at the intermediate position in the modern Tibetan-Tai-Kadai/Austronesian genetic cline. This suggests that the formation of Tibetan-Burman groups involved a large-scale gene flow from lowland southern Chinese. The southern ancestry could be further modelled as deriving from Vietnam's Late Neolithic-related inland Southeast Asia agricultural populations and Taiwan's Iron Age-related coastal rice-farming populations. Compared to the Tibeto-Burman speakers in the Tibetan-Yi Corridor reported previously, the Tibeto-Burman groups in the Guizhou region received additional gene flow from the southeast coastal area of China. We show a difference between the genetic profiles of the Tibeto-Burman speakers of the Tibetan-Yi Corridor and the Guizhou province. Vast mountain ranges and rivers in Southwest China may have decelerated the westward expansion of the southeast coastal East Asians. Our results demonstrate the complex genetic profile in the Guizhou region in Southwest China and support the multiple waves of human migration in the southern area of East Asia.

Ancient genomes reveal millet farming-related demic diffusion from the Yellow River into southwest China.

The study of southwest China is vital for understanding the dispersal and development of farming because of the coexistence of millet and rice in this region since the Neolithic period.1,2 However, the process of the Neolithic transition in southwest China is largely unknown, mainly due to the lack of ancient DNA from the Neolithic period. Here, we report genome-wide data from 11 human samples from the Gaoshan and Haimenkou sites with mixed farming of millet and rice dating to between 4,500 and 3,000 years before present in southwest China. The two ancient groups derived approximately 90% of their ancestry from the Neolithic Yellow River farmers, suggesting a demic diffusion of millet farming to southwest China. We inferred their remaining ancestry to be derived from a Hòabìnhian-related hunter-gatherer lineage. We did not detect rice farmer-related ancestry in the two ancient groups, which indicates that they likely adopted rice farming without genetic assimilation. We, however, observed rice farmer-related ancestry in the formation of some present-day Tibeto-Burman populations. Our results suggested the occurrence of both demic and cultural diffusion in the development of Neolithic mixed farming in some parts of southwest China.

Inferring the population history of Kyrgyz in Xinjiang, Northwest China from genome-wide array genotyping.

OBJECTIVES: Xinjiang plays a vital role in the trans-Eurasian population migration, language diffusion, and culture and technology exchange. However, the underrepresentation of Xinjiang's genomes has hindered a more comprehensive understanding of Xinjiang's genetic structure and population history. MATERIALS AND METHODS: We collected and genotyped 70 southern Xinjiang's Kyrgyz (SXJK) individuals and combined the data with modern and ancient Eurasians published. We used allele-frequency methods, including PCA, ADMIXTURE, f-statistics, qpWave/qpAdm, ALDER, Treemix, and haplotype-shared methods including shared-IBD segments, fineSTRUCTURE, and GLOBETROTTER to unveil the fine-scale population structure and reconstruct admixture history. RESULTS: We identified genetic substructure within the SXJK population with subgroups showing different genetic affinities to West and East Eurasians. All SXJK subgroups were suggested to have close genetic relationships with surrounding Turkic-speaking groups that is, Uyghur, Kyrgyz from north Xinjiang and Tajikistan, and Chinese Kazakh, suggesting a shared ancestry among those populations. Outgroup-f3 and symmetrical f4 statistics showed a high genetic affinity of SXJK to present-day Tungusic, Mongolic-speaking populations and Ancient Northeast Asian (ANA) related groups. Allele sharing and haplotype sharing profiles revealed the east-west admixture pattern of SXJK. The qpAdm-based admixture models showed that SXJK derived ancestry from East Eurasian (ANA and East Asian, 42.7%-83.3%) and West Eurasian (Western Steppe herders and Central Asian, 16.7%-57.3%), the recent east-west admixture event could be traced to 1000 years ago based on ALDER and GLOBETROTTER analysis. DISCUSSION: The high genetic affinity of SXJK to present-day Tungusic and Mongolic-speaking populations and short-shared IBD segments indicated their shared common ancestry. SXJK harbored a close genetic affinity to ANA-related populations, indicating the Northeast Asian origin of SXJK. The West and East Eurasian admixture models observed in SXJK further provided evidence of the dynamic admixture history in Xinjiang. The east-west admixture pattern and the identified ancestral makeup of SXJK suggested a genetic continuity from some Iron Age Xinjiang populations to present-day SXJK.

Toxicity and ecological risk assessment for two AhR agonistic pesticides mepanipyrim and cyprodinil and their metabolites.

Mepanipyrim and cyprodinil are widely used to control and/or prevent fungal diseases in fruit culture. They are frequently detected in the aquatic environment and some food commodities. Different from TCDD, mepanipyrim and cyprodinil are more easily metabolised in the environments. However, the risk of their metabolites to the ecological environment is unclear and needs to be further confirmed. In this study, we investigated the temporal pattern of mepanipyrim- and cyprodinil-induced CYP1A and AhR2 expression and EROD enzyme activity at different time frames during zebrafish embryonic and larval development. Then, we assessed the ecological risk of mepanipyrim, cyprodinil, and their metabolites to aquatic organisms. Our results showed that mepanipyrim and cyprodinil exposure could increase the expression level of cyp1a and ahr2 genes and EROD activity by a dynamic pattern in different developmental stages of zebrafish. Besides, their several metabolites showed strong AhR agonistic activity. Importantly, these metabolites could cause potential ecological risks to aquatic organisms and should be paid more attention to. Our results would provide an important reference value for environmental pollution control and the use management of mepanipyrim and cyprodinil.

The genetic structure and admixture of Manchus and Koreans in northeast China.

BACKGROUND: The fine-scale genetic profiles and population history of Manchus and Koreans remain unclear. AIM: To infer a fine-scale genetic structure and admixture of Manchu and Korean populations. SUBJECTS AND METHODS: We collected and genotyped 16 Manchus from Liaoning and 18 Koreans from Jilin province with about 700K genome-wide SNPs. We analysed the data using principal component analysis (PCA), ADMIXTURE, Fst, TreeMix, f-statistics, qpWave, and qpAdm. RESULTS: Manchus and Koreans showed a genetic affinity with northern East Asians. Chinese Koreans showed a long-term genetic continuity with Bronze Age populations from the West Liao River and had a strong affinity with Koreans in South Korea and Japan. Manchus had a different genetic profile compared with other Tungusic populations since the Manchus received additional genetic influence from the southern Chinese but didn't have West Eurasian-related admixture. CONCLUSIONS: The genetic formation of Manchus involving southern Chinese was consistent with the extensive interactions between Manchus and populations from central and southern China. The large-scale genetic continuity between ancient West Liao River farmers and Koreans highlighted the role farming expansion played in the peopling of the Korean Peninsula.

Mepanipyrim induces cardiotoxicity of zebrafish (Danio rerio) larvae via promoting AhR-regulated COX expression pathway.

The wide use of pesticides has seriously threatened human health and the survival of beneficial organisms. The fungicide mepanipyrim is widely used in viticulture practices. Studies of mepanipyrim-induced toxicity in organisms are still scarce, especially studies on cardiotoxicity. In this study, we aimed to investigate mepanipyrim-induced cardiotoxicity in zebrafish (Danio rerio) larvae. We found that mepanipyrim could induce cardiotoxicity by altering the heart rate and cardiomyocyte diameter of larvae. Meanwhile, RNA sequencing and RT-qPCR data indicated that mepanipyrim exposure could dramatically alter the mRNA expression of calcium signaling pathway-, cardiac muscle contraction-, and oxidative respiratory chain-related genes. Interestingly, by the CALUX cell bioassay, we found that most cytochrome c oxidase (COX) family genes exhibited potential AhR-regulated activity, suggesting that mepanipyrim induced cardiotoxicity via a novel AhR-regulated manner in larvae. Additionally, the AhR antagonist CH223191 could effectively prevent mepanipyrim-induced cardiotoxicity in zebrafish larvae. In conclusion, the AhR agonist mepanipyrim could induce cardiotoxicity in a novel unreported AhR-regulated manner, which could specifically affect the expression of COX family genes involved in the mitochondrial oxidative respiratory chain. Our data will help explain the toxic effects of mepanipyrim on organisms and provide new insight into the AhR agonistic activity pesticide-induced cardiotoxicity.

Cultural and demic co-diffusion of Tubo Empire on Tibetan Plateau.

A high point of Tibetan Plateau (TP) civilization, the expansive Tubo Empire (618-842 AD) wielded great influence across ancient western China. However, whether the Tubo expansion was cultural or demic remains unclear due to sparse ancient DNA sampling. Here, we reported ten ancient genomes at 0.017- to 0.867-fold coverages from the Dulan site with typical Tubo archaeological culture dating to 1308-1130 BP. Nine individuals from three different grave types have close relationship with previously reported ancient highlanders from the southwestern Himalayas and modern core-Tibetan populations. A Dulan-related Tubo ancestry contributed overwhelmingly (95%-100%) to the formation of modern Tibetans. A genetic outlier with dominant Eurasian steppe-related ancestry suggesting a potential population movement into the Tubo-controlled regions from Central Asia. Together with archeological evidence from burial styles and customs, our study suggested the impact of the Tubo empire on the northeast edge of the TP involved both cultural and demic diffusion.

An overlooked source of nanosized lead particles in the atmosphere: Residential honeycomb briquette combustion.

Atmospheric lead (Pb) pollution has attracted long-term and widespread concerns due to its high toxicity. The definite source identification of atmospheric Pb is the key step to mitigate this pollution. Here, we first report an overlooked source of atmospheric nanosized Pb particles using transmission electron microscopy and bulk sample analyses, finding that residential honeycomb briquette combustion emits large numbers of nanosized Pb-rich particles. We found that 33.7 ± 19.9 % of primary particles by number from residential honeycomb briquette combustion contains the crystalline Pb particles. These Pb-rich particles range in size from 14 to 956 nm with a mean diameter of 117 nm. Compared with raw coal chunks, honeycomb briquette combustion could emit less carbonaceous particles, but largely increase nanosized Pb particle emissions. This result is attributed to two key factors: (1) higher Pb content in honeycomb briquette (63.6 µg g-1) than that in coal chunk (8.5 µg g-1), and (2) higher Pb release rate for honeycomb briquette (62.3 %) caused by honeycomb structure than that for coal chunk (20.1 %). This study highlights that atmospheric and health implications of high emissions of toxic nanosized Pb from honeycomb briquette should be paid more attention in future research on ambient and indoor airs.

Aryl hydrocarbon receptor agonist diuron and its metabolites cause reproductive disorders in male marine medaka (Oryzias melastigma).

Diuron, a widely used phenylurea herbicide, has been frequently detected in marine organism and seawater all over the world. But the understanding of potential damage of diuron on reproduction in marine fish is currently not enough. Herein, marine medaka (Oryzias melastigma) were continuously exposed to 0, 5, 50, 500, and 5000 ng/L diuron from embryo (0 dpf) to adult (180 dpf) stage. The results suggested that diuron had an adverse influence on male reproduction for marine medaka, including decreased gonado somatic index, histological changes of testes, decreased mobility of sperm, and reduced fecundity through disrupting the balance of sex hormone and genes expression related to hypothalamus-pituitary-gonadal-liver (HPGL) axis. The reduced fecundity was reflected in abnormal sexual behaviors, further inhibited growth and development of F1 embryo and larvae. Moreover, the proportion of diuron metabolites (DCPMU and DCPU) was increased in fish, but the proportion of diuron was decreased with the increasing of exposure concentration. Diuron, DCPMU, and DCPU was identified as aryl hydrocarbon receptor agonist (AhR) agonist using in silico and in vivo models. DCPMU and DCPU induced the gene expression of AhR signaling and metabolizing enzymes (such as cyp1a1) in the livers. A great deal of major metabolites affected various organs related to HPGL axis of male marine medaka and led to serious reproductive disorders. Consequently, it reveals that long-term exposure to environmentally relevant concentrations of diuron and even AhR agonist pesticides pose a potential ecological risk for marine fish.

Fine-Scale Population Admixture Landscape of Tai-Kadai-Speaking Maonan in Southwest China Inferred From Genome-Wide SNP Data.

Guizhou Province harbors extensive ethnolinguistic and cultural diversity with Sino-Tibetan-, Hmong-Mien-, and Tai-Kadai-speaking populations. However, previous genetic analyses mainly focused on the genetic admixture history of the former two linguistic groups. The admixture history of Tai-Kadai-speaking populations in Guizhou needed to be characterized further. Thus, we genotyped genome-wide SNP data from 41 Tai-Kadai-speaking Maonan people and made a comprehensive population genetic analysis to explore their genetic origin and admixture history based on the pattern of the sharing alleles and haplotypes. We found a genetic affinity among geographically different Tai-Kadai-speaking populations, especially for Guizhou Maonan people and reference Maonan from Guangxi. Furthermore, formal tests based on the f 3 /f 4 -statistics further identified an adjacent connection between Maonan and geographically adjacent Hmong-Mien and Sino-Tibetan people, which was consistent with their historically documented shared material culture (Zhang et al., iScience, 2020, 23, 101032). Fitted qpAdm-based two-way admixture models with ancestral sources from northern and southern East Asians demonstrated that Maonan people were an admixed population with primary ancestry related to Guangxi historical people and a minor proportion of ancestry from Northeast Asians, consistent with their linguistically supported southern China origin. Here, we presented the landscape of genetic structure and diversity of Maonan people and a simple demographic model for their evolutionary process. Further whole-genome-sequence-based projects can be presented with more detailed information about the population history and adaptative history of the Guizhou Maonan people.

Resveratrol ameliorates polycystic ovary syndrome via transzonal projections within oocyte-granulosa cell communication.

Rationale: Polycystic ovary syndrome (PCOS) is closely linked to follicular dysplasia and impaired bidirectional oocyte-granulosa cell (GC) communication. Given that PCOS is a heterogeneous, multifactorial endocrine disorder, it is important to clarify the pathophysiology of this ovarian disease and identify a specific treatment. Methods: We generated PCOS rat models based on neonatal tributyltin (TBT) exposure and studied the therapeutic effect and mechanism of resveratrol (RSV), a natural plant polyphenol. Transcriptome analysis was conducted to screen the significantly changed pathways, and a series of experiments, such as quantitative real-time polymerase chain reaction (PCR), Western blot and phalloidin staining, were performed in rat ovaries. We also observed similar changes in human PCOS samples using Gene Expression Omnibus (GEO) database analysis and quantitative real-time PCR. Results: We first found that injury to transzonal projections (TZPs), which are specialized filopodia that mediate oocyte-GC communication in follicles, may play an important role in the etiology of PCOS. We successfully established PCOS rat models using TBT and found that overexpressed calcium-/calmodulin-dependent protein kinase II beta (CaMKIIß) inhibited TZP assembly. In addition, TZP disruption and CAMK2B upregulation were also observed in samples from PCOS patients. Moreover, we demonstrated that RSV potently ameliorated ovarian failure and estrus cycle disorder through TZP recovery via increased cytoplasmic calcium levels and excessive phosphorylation of CaMKIIß. Conclusions: Our data indicated that upregulation of CaMKIIß may play a critical role in regulating TZP assembly and may be involved in the pathogenesis of PCOS associated with ovarian dysfunction. Investigation of TZPs and RSV as potent CaMKIIß activators provides new insight and a therapeutic target for PCOS, which is helpful for improving female reproduction.

Genomic Insights Into the Genetic Structure and Natural Selection of Mongolians.

Mongolians dwell at the Eastern Eurasian Steppe, where is the agriculture and pasture interlaced area, practice pastoral subsistence strategies for generations, and have their own complex genetic formation history. There is evidence that the eastward expansion of Western Steppe herders transformed the lifestyle of post-Bronze Age Mongolia Plateau populations and brought gene flow into the gene pool of Eastern Eurasians. Here, we reported genome-wide data for 42 individuals from the Inner Mongolia Autonomous Region of North China. We observed that our studied Mongolians were structured into three distinct genetic clusters possessing different genetic affinity with previous studied Inner Mongolians and Mongols and various Eastern and Western Eurasian ancestries: two subgroups harbored dominant Eastern Eurasian ancestry from Neolithic millet farmers of Yellow River Basin; another subgroup derived Eastern Eurasian ancestry primarily from Neolithic hunter-gatherers of North Asia. Besides, three-way/four-way qpAdm admixture models revealed that both north and southern Western Eurasian ancestry related to the Western Steppe herders and Iranian farmers contributed to the genetic materials into modern Mongolians. ALDER-based admixture coefficient and haplotype-based GLOBETROTTER demonstrated that the former western ancestry detected in modern Mongolian could be recently traced back to a historic period in accordance with the historical record about the westward expansion of the Mongol empire. Furthermore, the natural selection analysis of Mongolians showed that the Major Histocompatibility Complex (MHC) region underwent significantly positive selective sweeps. The functional genes, alcohol dehydrogenase (ADH) and lactase persistence (LCT), were not identified, while the higher/lower frequencies of derived mutations were strongly correlated with the genetic affinity to East Asian/Western Eurasian populations. Our attested complex population movement and admixture in the agriculture and pasture interlaced area played an important role in the formation of modern Mongolians.

Comparative Performance of the MGISEQ-2000 and Illumina X-Ten Sequencing Platforms for Paleogenomics.

The MGISEQ-2000 sequencer is widely used in various omics studies, but the performance of this platform for paleogenomics has not been evaluated. We here compare the performance of MGISEQ-2000 with the Illumina X-Ten on ancient human DNA using four samples from 1750BCE to 60CE. We found there were only slight differences between the two platforms in most parameters (duplication rate, sequencing bias, θ, δS, and λ). MGISEQ-2000 performed well on endogenous rate and library complexity although X-Ten had a higher average base quality and lower error rate. Our results suggest that MGISEQ-2000 and X-Ten have comparable performance, and MGISEQ-2000 can be an alternative platform for paleogenomics sequencing.

Genomic Insight Into the Population Structure and Admixture History of Tai-Kadai-Speaking Sui People in Southwest China.

Sui people, which belong to the Tai-Kadai-speaking family, remain poorly characterized due to a lack of genome-wide data. To infer the fine-scale population genetic structure and putative genetic sources of the Sui people, we genotyped 498,655 genome-wide single-nucleotide polymorphisms (SNPs) using SNP arrays in 68 Sui individuals from seven indigenous populations in Guizhou province and Guangxi Zhuang Autonomous Region in Southwest China and co-analyzed with available East Asians via a series of population genetic methods including principal component analysis (PCA), ADMIXTURE, pairwise Fst genetic distance, f-statistics, qpWave, and qpAdm. Our results revealed that Guangxi and Guizhou Sui people showed a strong genetic affinity with populations from southern China and Southeast Asia, especially Tai-Kadai- and Hmong-Mien-speaking populations as well as ancient Iron Age Taiwan Hanben, Gongguan individuals supporting the hypothesis that Sui people came from southern China originally. The indigenous Tai-Kadai-related ancestry (represented by Li), Northern East Asian-related ancestry, and Hmong-Mien-related lineage contributed to the formation processes of the Sui people. We identified the genetic substructure within Sui groups: Guizhou Sui people were relatively homogeneous and possessed similar genetic profiles with neighboring Tai-Kadai-related populations, such as Maonan. While Sui people in Yizhou and Huanjiang of Guangxi might receive unique, additional gene flow from Hmong-Mien-speaking populations and Northern East Asians, respectively, after the divergence within other Sui populations. Sui people could be modeled as the admixture of ancient Yellow River Basin farmer-related ancestry (36.2-54.7%) and ancient coastal Southeast Asian-related ancestry (45.3-63.8%). We also identified the potential positive selection signals related to the disease susceptibility in Sui people via integrated haplotype score (iHS) and number of segregating sites by length (nSL) scores. These genomic findings provided new insights into the demographic history of Tai-Kadai-speaking Sui people and their interaction with neighboring populations in Southern China.

Genomic Insights Into the Admixture History of Mongolic- and Tungusic-Speaking Populations From Southwestern East Asia.

As a major part of the modern Trans-Eurasian or Altaic language family, most of the Mongolic and Tungusic languages were mainly spoken in northern China, Mongolia, and southern Siberia, but some were also found in southern China. Previous genetic surveys only focused on the dissection of genetic structure of northern Altaic-speaking populations; however, the ancestral origin and genomic diversification of Mongolic and Tungusic-speaking populations from southwestern East Asia remain poorly understood because of the paucity of high-density sampling and genome-wide data. Here, we generated genome-wide data at nearly 700,000 single-nucleotide polymorphisms (SNPs) in 26 Mongolians and 55 Manchus collected from Guizhou province in southwestern China. We applied principal component analysis (PCA), ADMIXTURE, f statistics, qpWave/qpAdm analysis, qpGraph, TreeMix, Fst, and ALDER to infer the fine-scale population genetic structure and admixture history. We found significant genetic differentiation between northern and southern Mongolic and Tungusic speakers, as one specific genetic cline of Manchu and Mongolian was identified in Guizhou province. Further results from ADMIXTURE and f statistics showed that the studied Guizhou Mongolians and Manchus had a strong genetic affinity with southern East Asians, especially for inland southern East Asians. The qpAdm-based estimates of ancestry admixture proportion demonstrated that Guizhou Mongolians and Manchus people could be modeled as the admixtures of one northern ancestry related to northern Tungusic/Mongolic speakers or Yellow River farmers and one southern ancestry associated with Austronesian, Tai-Kadai, and Austroasiatic speakers. The qpGraph-based phylogeny and neighbor-joining tree further confirmed that Guizhou Manchus and Mongolians derived approximately half of the ancestry from their northern ancestors and the other half from southern Indigenous East Asians. The estimated admixture time ranged from 600 to 1,000 years ago, which further confirmed the admixture events were mediated via the Mongolians Empire expansion during the formation of the Yuan dynasty.

Screening of potential oestrogen receptor α agonists in pesticides via in silico, in vitro and in vivo methods.

In modern agricultural management, the use of pesticides is indispensable. Due to their massive use worldwide, pesticides represent a latent risk to both humans and the environment. In the present study, 1056 frequently used pesticides were screened for oestrogen receptor (ER) agonistic activity by using in silico methods. We found that 72 and 47 pesticides potentially have ER agonistic activity by the machine learning methods random forest (RF) and deep neural network (DNN), respectively. Among endocrine-disrupting chemicals (EDCs), 14 have been reported as EDCs or ER agonists by previous studies. We selected 3 reported and 7 previously unreported pesticides from 76 potential ER agonists to further assess ERα agonistic activity. All 10 selected pesticides exhibited ERα agonistic activity in human cells or zebrafish. In the dual-luciferase reporter gene assays, six pesticides exhibited ERα agonistic activity. Additionally, nine pesticides could induce mRNA expression of the pS2 and NRF1 genes in MCF-7 cells, and seven pesticides could induce mRNA expression of the vtg1 and vtg2 genes in zebrafish. Importantly, the remaining 48 out of 76 potential ER agonists, none of which have previously been reported to have endocrine-disrupting effects or oestrogenic activity, should be of great concern. Our screening results can inform environmental protection goals and play an important role in environmental protection and early warnings to human health.

Improvement in the screening performance of potential aryl hydrocarbon receptor ligands by using supervised machine learning.

The aryl hydrocarbon receptor (AhR), which is a ligand-dependent transcription factor, plays a crucial role in the regulation of xenobiotic metabolism. There are a large number of artificial or natural molecules in the environment that can activate AhR. In this study, we developed a virtual screening procedure to identify potential ligands of AhR. One structure-based method and two ligand-based methods were used for the virtual screening procedure. The results showed that the precision rate (0.96) and recall rate (0.64) of our procedure were significantly higher than those of a procedure used in a previous study, which suggests that supervised machine learning techniques can greatly improve the performance of virtual screening. Moreover, a pesticide dataset including 777 frequently used pesticides was screened. Seventy-seven pesticides were identified as potential AhR ligands by all three screening methods, among which 12 have never been previously reported as AhR agonists. Two non-agonist AhR ligands and 14 of the 77 pesticides were randomly selected for testing by in vitro and in vivo assays. All 14 pesticides showed different degrees of AhR agonistic activity, and none of the two non-agonist AhR ligand pesticides showed AhR agonistic activity, which suggests that our procedure had good robustness. Four of the pesticides were reported as AhR agonists for the first time, suggesting that these pesticides may need further toxicity assessment. In general, our procedure is a rapid, powerful and computationally inexpensive tool for predicting chemicals with AhR agonistic activity, which could be useful for environmental risk prediction and management.

Exposure to the AhR agonist cyprodinil impacts the cardiac development and function of zebrafish larvae.

Cyprodinil is a broad-spectrum pyrimidine amine fungicide that has been reportedly used worldwide. However, toxicity studies of cyprodinil on aquatic organisms, specifically zebrafish (Danio rerio), are lacking. In our present study, we predicted cyprodinil binding to the aryl hydrocarbon receptor (AhR) by using molecular docking simulation. Then, we used recombinant HepG2 cells and Tg(cyp1a1-12DRE:egfp) transgenic zebrafish to further assess the AhR agonistic activity of cyprodinil. Besides, the significant upregulation of cyp1a1 further verified that statement. Moreover, we found that zebrafish exposure to cyprodinil induced developmental toxicity in the larvae, particularly during cardiac development. The expression levels of cardiac development-related genes, namely tbx5, nkx2.5, gata4, and tnnt2, were markedly altered, which might cause the adverse effects of cyprodinil on cardiac function and development. In summary, we found that cyprodinil, as an AhR agonist, induced development toxicity in zebrafish larvae, especially on cardiac. Data here can assess the potential effects on organisms in the aquatic environment and promote the regulation and safe use of cyprodinil.