Integrating ultra-high-performance liquid chromatography tandem mass spectrometry and imaged capillary isoelectric focusing for in-depth characterization of complex fusion proteins.

RATIONALE: Fc-fusion proteins represent a successful class of biopharmaceutical products, which combine the tailored pharmacological properties of biological ligands with the multiple functions of the fragment crystallizable domain of immunoglobulins. There is great diversity in terms of possible biological ligands creating highly diverse structures, therefore the analytical characterization of fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. METHODS: Employing etanercept analogues as studied fusion proteins, the Orbitrap mass analyzer with ultra-high performance liquid chromatography (UHPLC-MS) and imaged capillary isoelectric focusing (icIEF) were utilized for the in-depth fusion protein characterization. RESULTS: The amino acid sequence coverage, peptide mapping, and post-translational modifications of etanercept analogues were analyzed by UHPLC-MS. The post-translational modification results were complemented by imaged capillary isoelectric focusing to produce quality research on etanercept analogues. CONCLUSIONS: The developed workflow integrating UHPLC-MS and icIEF provided an innovative strategy for characterizing complex fusion proteins in the process of quality control and manufacturing.

Using Machine Learning to Predict Adverse Effects of Metallic Nanomaterials to Various Aquatic Organisms.

The wide production and use of metallic nanomaterials (MNMs) leads to increased emissions into the aquatic environments and induces high potential risks. Experimentally evaluating the (eco)toxicity of MNMs is time-consuming and expensive due to the multiple environmental factors, the complexity of material properties, and the species diversity. Machine learning (ML) models provide an option to deal with heterogeneous data sets and complex relationships. The present study established an in silico model based on a machine learning properties-environmental conditions-multi species-toxicity prediction model (ML-PEMST) that can be applied to predict the toxicity of different MNMs toward multiple aquatic species. Feature importance and interaction analysis based on the random forest method indicated that exposure duration, illumination, primary size, and hydrodynamic diameter were the main factors affecting the ecotoxicity of MNMs to a variety of aquatic organisms. Illumination was demonstrated to have the most interaction with the other features. Moreover, incorporating additional detailed information on the ecological traits of the test species will allow us to further optimize and improve the predictive performance of the model. This study provides a new approach for ecotoxicity predictions for organisms in the aquatic environment and will help us to further explore exposure pathways and the risk assessment of MNMs.

[Application of automatic injection device based on automatic hemostasis in injection of radiopharmaceutical bolus injection].

In clinical practice, radiopharmaceutical dynamic imaging technology requires the bolus injection method to complete injection. Due to the failure rate and radiation damage of manual injection, even experienced technicians still bear a lot of psychological burden. This study combined the advantages and disadvantages of various manual injection modes to develop the radiopharmaceutical bolus injector, and explored the application of automatic injection in the field of bolus injection from four aspects: radiation protection, occlusion response, sterility of injection process and effect of bolus injection. Compared with the current mainstream manual injection method, the bolus manufactured by the radiopharmaceutical bolus injector based on the automatic hemostasis method had a narrower full width at half maximum and better repeatability. At the same time, radiopharmaceutical bolus injector had reduced the radiation dose of the technician's palm by 98.8%, and ensured more efficient vein occlusion recognition performance and sterility of the entire injection process. The radiopharmaceutical bolus injector based on automatic hemostasis has application potential in improving the effect and repeatability of bolus injection.

Production of Reactive Oxygen Species via Nanobubble Water Improves Radish Seed Water Absorption and the Expression of Aquaporin Genes.

Nanobubbles (NBs) stimulate seed germination; however, the mechanism of the promotion effect of NBs remains unclear. The impact of NBs on seed water absorption was investigated; we subsequently studied the genes associated with the response of radish seeds to NB water and used RNA sequencing to generate their expression profiles, especially those of aquaporin genes. NB water significantly promoted germination. The times at which 50% of the germinating seeds achieved germination (T50) for the submerged radish seeds in NB and control water were 11.6 and 17.4 h, respectively. NB water-germinated radish seeds showed a water uptake rate coefficient that was 15% higher than that of those germinated in control water. Through GO enrichment and cluster analyses, it was evident that NB water significantly increased the level of expression of the genes associated with the following activities: oxidoreductase, peroxidase, and antioxidant. Our results demonstrated that NB water increases the water uptake rate of radish seeds via two mechanisms. The NB water-produced exogenous hydroxyl radical (•OH) increases the seed coat's water permeability and enhances cell wall loosening, and NB water increases the aquaporin gene expression level of radish seeds.

Enhanced Removal of Free Radicals by Aqueous Hydrogen Nanobubbles and Their Role in Oxidative Stress.

Elevated levels of reactive oxygen radicals caused by environmental stress are the key triggers of inflammation, aging, and disease; thus, it is critical to develop novel reactive oxygen radical scavenging methods with high efficiency and low toxicity. As a result of their selective reactive oxygen radical removal, hydrogen molecules are strong candidates, but their application is limited by the small hydrogen supply and short duration of action. In this study, we for the first time combined nanobubble (NB) technology and hydrogen water to remove reactive oxygen species (ROS) using copper ions as a representative environmental pollutant and Tetrahymena thermophila as a model organism. Hydrogen NBs displayed a remarkable capability of removing H2O2 and O2•- at molar ratios of 8:1 and 240:1, respectively, which were unable to be removed by dissolved hydrogen molecules only. During the oxidative defense phase, hydrogen NB water either directly removed ROS or increased the activity and relative expression of glutathione peroxidase (GSH-Px). During the oxidative inhibition phase, hydrogen NB water exerted antioxidant effects mainly by increasing the activities of superoxide dismutase and GSH-Px as well as the expression of the corresponding genes. Our results provide an important theoretical support for the wide application of hydrogen NBs in empowering the antioxidant defense system.

Global Exposure to Per- and Polyfluoroalkyl Substances and Associated Burden of Low Birthweight.

Low birthweight (LBW) is a worldwide public health concern, while the global burden of LBW attributable to endocrine-disrupting chemicals, such as per- and polyfluoroalkyl substances (PFAS), has not yet been evaluated. Here, we established a large dataset for the biomonitoring of seven representative congeners of PFAS by examining data from 2325 publications. Global exposure to perfluorooctanesulfonic acid (PFOS) was the highest, followed by perfluorohexanesulfonic acid (PFHxS) and perfluorooctanoic acid (PFOA). Spatiotemporal exposure to PFAS varied considerably, with daily intake estimated in the range of 0.01-1.7 ng/kg/day. Moreover, decreasing trends in PFOS, PFHxS, and PFOA exposure were noted in most regions of the world over the past two decades, but such trends were not observed for other PFAS with long carbon chains, especially in East Asia. Furthermore, we estimated that human exposure to PFOA contributed to approximately 461,635 (95% confidence interval: 57,418 to 854,645) cases per year of LBW during the past two decades, predominantly from Asian regions. Although our estimation may be constrained by uncertainties from the dose-response curve and data availability, this study has unveiled that PFAS might be a contributor to global LBW prevalence during 2000-2019, supporting continuous actions to mitigate PFAS contamination.

Effects of water quality and bacterial community composition on dissolved organic matter structure in Daihai lake and the mechanisms.

The migration of organic matter in salinized lakes was critical in maintaining ecological balance and material circulation process of inland shallow lakes. To clarify the ecological and microbial mechanism of material transport and transformation, the microbial community structure and the characteristics of dissolved organic matter (DOM) in the sediment of Daihai Lake, a typical saline lake at the Yellow River Basin, were explored with three-dimensional excitation and emission matrix fluorescence (3DEEM), parallel factor analysis (PARAFAC) and 16 S rRNA techniques. The correlation between environmental factors, DOM composition and the bacterial community structure were also studied for identifying the key factors of community formation. DOM in the lake demonstrated both terrigenous and endogenous characteristics. Protein-like materials accounted for 74% of the total fluorescence intensity in the sediment, where 1127 species, 671 genera, 468 families, 157 classes, 317 orders, 59 phyla of microorganisms were detected. Among the top 10 abundant taxa of each level, Firmicutes, Actinobacterota, Acidimicrobiia and Alphaproteobacteria had the greatest influence on the composition and structure of DOM (|R| > 0.7, p < 0.01). Microbial metabolism was a key process of transforming sediment organic matter from terrestrial humic-like to protein-like matter, accounting for 81% of total fluorescence signal in saline lake samples, while salinity, temperature, dissolved oxygen and electrical conductivity also had significant impacts during the process (|R|>0.7, p < 0.05). The research provides fundamental data and enlightenment for the improvement of the saline inland lake environment.

Mortality Risk Associated with Short-Term Exposure to Particulate Matter in China: Estimating Error and Implication.

Most previous studies used a specific size of particulate matter (PMx) for dosimetry estimation when determining particulate matter (PM)-associated risk, which precluded the impact of other sizes of PM. Here, we used a multiple-path particle dosimetry model to determine the deposition of PM in human airways and further estimated the associated mortality risk in 205 cities in China. Results showed that the fractions of PM1, PM1-2.5, and coarse PM (PM2.5-10) deposited in the tracheobronchial (TB) and pulmonary airways were estimated in ranges of 11.06-12.83, 19.9-26.37, and 5.35-9.81%, respectively. Each 10 µg/m3 increase in deposited PM was significantly associated with a nationwide increment of 1.12% (95% confidence interval, CI, 0.77-1.49%) for total nonaccidental mortality. Short-term exposure to PM during 2014-2017 resulted in a nationwide mortality of 98 826 cases/year, with contributions from PM1, PM1-2.5, coarse PM of 37.7, 43.1, and 19.2%, respectively. Our study demonstrated that the estimated mortality counts may be associated with the coefficient of variation of dosimetry estimations. In addition, we revealed the caution should be exercised when interpreting PMx-associated risk and further reinforced the importance of size distribution in relevant research.

Effect of different shapes of Nano-Cu2O and humic acid on two-generations of Daphnia Magna.

Nanomaterials (NMs) have significant technological advantages due to their unique properties but their release into the environment also carry potential eco-toxicological risks. Cu2O NMs have numerous industrial and commercial applications, however less is known about the risks to human health and ecosystems. This study investigated different shapes of nano-Cu2O (cubic and octahedron) with humic acid (HA: 2 mg/L and 10 mg/L) on the reproduction, growth, survival rate and Cu accumulation in the two-generations of Daphnia Magna species. Nano-Cu2O cubic and octahedron shapes were analyzed by SEM, demonstrating {111} and {100} facets. The results indicated that octahedron has the more evident toxic effect than cubic shape for both generations. Continuous exposure to nano-Cu2O exhibited negative effects on body length, cumulative neonates and Cu accumulation in filial generation F (1). Survival rate of Cu2O octahedron was the least 66%, while Cu2O cubic was 73% for parental generation F (0). In addition, HA showed a significant reduction in the toxicity of nano-Cu2O for both generations, leading to an enhanced survival rate, body length and reproduction. This study provides insights of the long-term effect of different shapes of nano-Cu2O in the existence of humic acid to the multi-generations ecosystem.

Influence of Humic Acid on Oxidative Stress Induced by Arsenite and Arsenate Waterborne Exposure in Danio rerio.

The studies on how humic acid (HA) influences the oxidative stress of arsenic in aquatic organism is limited. Using Danio rerio as case study, we explored the oxidative stress effects in aquatic organism after 96 h exposure to the HA and arsenic. Results revealed the co-exposure of HA and arsenite elevated the superoxide dismutase activities and downgraded the malondialdehyde. Thus, we speculate that HA may alleviate the oxidative stress induced by arsenite, which may be caused by the HA's coating in combination with the complexation of arsenite and HA. In addition, HA acted as the reactive oxygen species scavenger, promising to eliminate the oxygen free radicals. Contrastingly, HA may impact little on the arsenate exposure. This study can help better understand oxidative stress mechanism of co-exposure of arsenic and HA in aquatic environment.

Characterizing the interactions between copper ions and dissolved organic matter using fluorescence excitation-emission matrices with two-dimensional Savitzky-Golay second-order differentiation.

Monitoring dissolved organic matter (DOM) content in aquatic environments is crucial for not only understanding the dynamics of heavy metals but also predicting their bioavailability. Fluorescence spectroscopy is typically employed to characterise DOM. Here, the interaction between DOM and trace metals was investigated by combining excitation-emission matrix (EEM) quenching with two-dimensional Savitzky-Golay second-order differentiation (2D-SG-2nd-df) analysis. The 2D-SG-2nd-df analysis decomposed the EEM spectra of commercial humic acid (HA) samples into six separate fluorescence peaks, which agreed with the results obtained through conventional parallel factor (PARAFAC) analysis. Compared with PARAFAC modeling, the 2D-SG-2nd-df approach provided more valid and reliable results when the dataset contained distinct samples. Moreover, since the results obtained from 2D-SG-2nd-df for each sample are independent, shifts in the peak wavelength can be reproduced more efficiently using this method. Triplicate titration experiments showed clear differences in HA-copper interactions for samples with different HA composition and molecular weight. The binding strength between copper and low-molecular-weight DOM in water was weaker than that observed for high-molecular-weight DOM. The results obtained in this study will serve as a basis for applying 2D-SG-2nd-df not only to DOM but also to other samples studied using EEM measurements.

Validation of single nucleotide polymorphisms in invasive aspergillosis following hematopoietic cell transplantation.

Invasive aspergillosis (IA) is a significant cause of morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Previous studies have reported an association between IA development and single nucleotide polymorphisms (SNPs), but many SNPs have not been replicated in a separate cohort. The presence of a positive serum galactomannan assay (SGM+) has also been associated with a worse prognosis in patients with IA, and genetic determinants in this subset of patients have not been systematically studied. The study cohort included 2609 HCT recipients and their donor pairs: 483 with proven/probable IA (183 SGM+) and 2126 with no IA by standard criteria. Of 25 SNPs previously published, we analyzed 20 in 14 genes that passed quality control. Samples were genotyped via microarray, and SNPs that could not be genotyped were imputed. The primary aim was to replicate SNPs associated with proven/probable IA at 2 years; secondary goals were to explore the associations using an end point of SGM+ IA or proven/probable IA using a different genetic model or time to IA (3 months vs 2 years) compared with the original study. Two SNPs in 2 genes (PTX3, CLEC7a) were replicated. Thirteen SNPs in 9 genes had an association at P ≤ .05 using the secondary aims (PTX3, CLEC7a, CD209, CXCL10, TLR6, S100B, IFNG, PLG, TNFR1), with hazard ratios ranging from 1.2 to 3.29. Underlying genetic differences can influence development of IA following HCT. Identification of genetic predispositions to IA could have important implications in donor screening, risk stratification of recipients, monitoring, and prophylaxis.

Effect of chronic toxicity of the crystalline forms of TiO2 nanoparticles on the physiological parameters of Daphnia magna with a focus on index correlation analysis.

The widespread use of titanium dioxide nanoparticles (NPs) and their inevitable release into aquatic environments have caused great concerns about their ecotoxicity. However, the chronic toxicity to TiO2 NPs of aquatic organisms has not been fully understood. In particular, research is lacking on the influence of the crystalline forms of TiO2 NPs on their mechanisms of toxicity. This study investigated the chronic toxicity (i.e., 21-day toxicity tests) of 5 types of TiO2 NPs with various percentages of crystalline forms on Daphnia magna. Results revealed that the crystalline form composed of 80% anatase and 20% rutile (i.e., the M1 form) had the highest energy band gap (i.e., Eg, the energy interval between the valence band edge and the conduction band edge) and caused maximal D. magna mortality compared with other crystalline forms. The crystalline form comprising 100% rutile (i.e., the R-S form) had the lowest Eg and exhibited a minimal effect on the physiological parameters of D. magna. Moreover, in a suitable environment without TiO2 NPs, D. magna progenies could recover to a normal physiological level (e.g., the mortalities of D. magna progenies were lower than those of parental D. magna that were exposed to TiO2 NPs at a concentration of 0.5 mg/L). Correlation analysis revealed that the body length, time of first brood, and number of neonates in the first brood of D. magna were negatively correlated with titanium accumulation in vivo. Furthermore, the indices of Ti accumulation and the product of Eg and Ti accumulation (i.e., Eg × Ti accumulation) were positively correlated (p < 0.05) with D. magna mortality, thus indicating that crystalline forms with a high Eg may cause severe toxicity to aquatic organisms at the same TiO2 bioaccumulation level.

Two-generational effects and recovery of arsenic and arsenate on Daphnia magna in the presence of nano-TiO2.

The toxicity of arsenic (As) can be influenced by many environmental factors. Among them, nanomaterials can adsorb arsenic and alter its bioavailability in organisms. However, the studies on long-term effects of arsenic in the presence of nanoparticles are limited. Thus, the 21-d effect of titanium dioxide nanoparticles (nano-TiO2) on chronic toxicity of arsenic (arsenate and arsenite) was investigated in two generations of Daphnia magna. The exposed concentration of nano-TiO2 was 1 mg/L and the concentration of As(Ⅲ) or As(Ⅴ) was 0.2 mg/L which was lower than the 48 h-NOEC (no observed effect concentration). The survival, body length, average number of offspring and time of first brood were determined. Our results indicated that the exposure to nano-TiO2 and As during the parental generation can affect the health of offspring. Nano-TiO2 was found to significantly alleviate the mortality and reproduction inhibition of As on D. magna, and the alleviation of As(Ⅴ) was more prominent than that of As(Ⅲ). It is likely that nano-TiO2 alters the metabolism and adsorption condition of arsenic in the gastrointestinal tract of D. magna. Overall, these results indicate that the increase of arsenic adsorption onto nano-TiO2 in the gut of D. magna could alleviate the toxicity of arsenic. Nonetheless, further research should be conducted to study the influence of arsenic on the multi-generations of aquatic organisms, especially when it is coexisted with other substances.

A Cell-Based Strategy for Bioactivity Determination of Long-Acting Fc-Fusion Recombinant Human Growth Hormone.

The long-acting growth hormone (LAGH) is a promising alternative biopharmaceutical to treat growth hormone (GH) deficiency in children, and it was developed using a variety of technologies by several pharmaceutical companies. Most LAGH preparations, such as Fc fusion protein, are currently undergoing preclinical study and clinical trials. Accurate determination of bioactivity is critical for the efficacy of quality control systems of LAGH. The current in vivo rat weight gain assays used to determine the bioactivity of recombinant human GH (rhGH) in pharmacopoeias are time-consuming, expensive, and imprecise, and there are no recommended bioassays for LAGH bioactivity in pharmacopoeias. Therefore, we developed a cell-based bioassay for bioactivity determination of therapeutic long-acting Fc-fusion recombinant human growth hormone (rhGH-Fc) based on the luciferase reporter gene system, which is involved in the full-length human GH receptor (hGHR) and the SG (SIE and GAS) response element. The established bioassay was comprehensively validated according to the International Council for Harmonization (ICH) Q2 (R1) guidelines and the Chinese Pharmacopoeia, and is highly precise, time-saving, simple, and robust. The validated bioassay could be qualified for bioactivity determination during the research, development, and manufacture of rhGH-Fc, and other LAGH formulations.

The Characterization of Dissolved Organic Matter in Reclaimed Water and Its Influence on Copper Toxicity.

Water samples were collected from five phases of treatment in a municipal sewage reclaimed water plant and the DOM was characterized. Results indicated that the components and properties of DOM varied notably with sequential treatments, such as the fluorescence intensity, the molecular weight and the total acidity. Meanwhile the accumulation of Cu in Daphnia magna was analyzed following exposure to samples spiked with 50 µg/L copper, which were decreased by the presence of DOM in those water samples. Furthermore, this study found significant associations between fluorescence intensity and Cu accumulation (r2 = 0.778, p < 0.05), while increased total acidity was found to enhance the unit total organic carbon-Cu accumulation (r2 = 0.979, p < 0.01). This study provides useful information on the safety and effective management of reclaimed water as a potential water resource.

Genetic risk factors for sclerotic graft-versus-host disease.

Sclerotic graft-versus-host disease (GVHD) is a distinctive phenotype of chronic GVHD after allogeneic hematopoietic cell transplantation, characterized by fibrosis of skin or fascia. Sclerotic GVHD has clinical and histopathological similarities with systemic sclerosis, an autoimmune disease whose risk is influenced by genetic polymorphisms. We examined 13 candidate single-nucleotide polymorphisms (SNPs) that have a well-documented association with systemic sclerosis to determine whether these SNPs are also associated with the risk of sclerotic GVHD. The study cohort included 847 consecutive patients who were diagnosed with chronic GVHD. Genotyping was performed using microarrays, followed by imputation of unobserved SNPs. The donor rs10516487 (BANK1: B-cell scaffold protein with ankyrin repeats 1) TT genotype was associated with lower risk of sclerotic GVHD (hazard ratio [HR], 0.43; 95% confidence interval [CI], 0.21-0.87; P = .02). Donor and recipient rs2056626 (CD247: T-cell receptor ζ subunit) GG or GT genotypes were associated with higher risk of sclerotic GVHD (HR, 1.57; 95% CI, 1.13-2.18; P = .007 and HR, 1.66; 95% CI, 1.19-2.32; P = .003, respectively). Donor and recipient rs987870 (5'-flanking region of HLA-DPA1) CC genotypes were associated with higher risk of sclerotic GVHD (HR, 2.50; 95% CI, 1.22-5.11; P = .01 and HR, 2.13; 95% CI, 1.00-4.54; P = .05, respectively). In further analyses, the recipient DPA1*01:03∼DPB1*04:01 haplotype and certain amino acid substitutions in the recipient P1 peptide-binding pocket of the HLA-DP heterodimer were associated with risk of sclerotic GVHD. Genetic components associated with systemic sclerosis are also associated with sclerotic GVHD. HLA-DP-mediated antigen presentation, T-cell response, and B-cell activation have important roles in the pathogenic mechanisms of both diseases.

Replication of associations between genetic polymorphisms and chronic graft-versus-host disease.

Previous studies have identified single-nucleotide polymorphisms (SNPs) associated with the risk of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation. The current study determined whether these associations could be replicated in large cohorts of donors and recipients. Each SNP was tested with cohorts of patients having the same donor type (HLA-matched related, unrelated, or both) reported in the original publication, and testing was limited to the same genome (recipient or donor) and genetic model (dominant, recessive, or allelic) reported in the original study. The 21 SNPs reported in this study represent 19 genes, and the analysis encompassed 22 SNP association tests. The hazard ratio (HR) point estimates and risk ratio point estimates corresponding to odds ratios in previous studies consistently fall outside the 95% confidence intervals of HR estimates in the current study. Despite the large size of the cohorts available for the current study, the 95% confidence intervals for most HRs did not exclude 1.0. Three SNPs representing CTLA4, HPSE, and IL1R1 showed evidence of association with the risk of chronic GVHD in unrelated donor-recipient pairs from 1 cohort, but none of these associations was replicated when tested in unrelated donor-recipient pairs from an independent cohort. Two SNPs representing CCR6 and FGFR1OP showed possible associations with the risk of chronic GVHD in related donor-recipient pairs but not in unrelated donor-recipient pairs. These results remain to be tested for replication in other cohorts of related donor-recipient pairs.

Aging Influences on the Biokinetics of Functional TiO2 Nanoparticles with Different Surface Chemistries in Daphnia magna.

Nanoparticles functionalized with various surface capping moieties are now widely used in different fields, thus there is a major need to understand the behavior and fate of these nanoparticles in the environment. The present study investigated the biokinetics of fresh titanium dioxide nanoparticles (TiO2 NPs) or TiO2 NPs aged under artificial sunlight (16 h light: 8 h dark) for 1, 3, and 5 days, respectively. Two commercial functionalized TiO2 NPs (with SiO2 coating or SiO2 and polydimethylsiloxane coating) were employed in this study. Dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), and contact angle (CA) measurements demonstrated that the surface properties had changed due to the degradation during aging. The biokinetic parameters including dissolved uptake and depuration rate constant as well as bioconcentration factors were calculated by a biokinetic model. All the biokinetic parameters were significantly dependent on the aging process. Further data analysis showed that the CA of the TiO2 NPs affected the uptake rate constant and the fast compartmental efflux, and both CA and hydrodynamic diameter affected the fast compartmental efflux. These results were due to the changes of corresponding indexes during the aging process. Our work highlighted the necessity of monitoring the physicochemical indexes of functionalized NPs during aging in evaluation of their environmental risks.

Development of multi-metal interaction model for Daphnia magna: Significance of metallothionein in cellular redistribution.

Despite the great progress made in metal-induced toxicity mechanisms, a critical knowledge gap still exists in predicting adverse effects of heavy metals on living organisms in the natural environment, particularly during exposure to multi-metals. In this study, a multi-metal interaction model of Daphnia manga was developed in an effort to provide reasonable explanations regarding the joint effects resulting from exposure to multi-metals. Metallothionein (MT), a widely used biomarker, was selected. In this model, MT was supposed to play the role of a crucial transfer protein rather than detoxifying protein. Therefore, competitive complexation of metals to MT could highly affect the cellular metal redistribution. Thus, competitive complexation of MT in D. magna with metals like Pb2+, Cd2+ and Cu2+ was qualitatively studied. The results suggested that Cd2+ had the highest affinity towards MT, followed by Pb2+ and Cu2+. On the other hand, the combination of MT with Cu2+ appeared to alter its structure which resulted in higher affinity towards Pb2+. Overall, the predicted bioaccumulation of metals under multi-metal exposure was consisted with earlier reported studies. This model provided an alternative angle for joint effect through a combination of kinetic process and internal interactions, which could help to develop future models predicting toxicity to multi-metal exposure.