Osteoblasts and bone formation

© 2007 Sociedade Portuguesa de ReumatologiaBone is constantly being remodelled in a dynamic process where osteoblasts are responsible for bone formation and osteoclasts for its resorption. Osteoblasts are specialized mesenchymal cells that undergo a process of maturation where genes like core-binding factor α1 (Cbfa1) and osterix (Osx) play a very important role. Moreover, it was found recently that the Wnt/β-catenin pathway plays a part on osteoblast differentiation and proliferation. In fact, mutations on some of the proteins involved in this pathway, like the low-density lipoprotein receptor related protein 5/6 (LRP5/6), lead to bone diseases. Osteoblasts have also a role in the regulation of bone resorption through receptor activator of nuclear factor-κB (RANK) ligand (RANKL), that links to its receptor, RANK, on the surface of pre-osteoclast cells, inducing their differentiation and fusion. On the other hand, osteoblasts secrete a soluble decoy receptor (osteoprotegerin, OPG) that blocks RANK/RANKL interaction by binding to RANKL and, thus, prevents osteoclast differentiation and activation. Therefore, the balance between RANKL and OPG determines the formation and activity of osteoclasts. Another factor that influences bone mass is leptin, a hormone produced by adipocytes that have a dual effect. It can act through the central nervous system and diminish osteoblasts activity, or can have an osteogenic effect by binding directly to its receptors on the surface of osteoblast cells.info:eu-repo/semantics/publishedVersio

Effect of inflammation on bone : biological, structural and mechanical behavior

Tese de doutoramento, Ciências Biomédicas (Ciências Biopatalógicas), Universidade de Lisboa, Faculdade de Medicina, 2011The term osteoimmunology was used for the first time in 2000 to describe the interaction of cells from the immune system and bone These two systems have several regulatory factors in common, such as cytokines, transcription factors and receptors. Consequently, they interact with each other both in physiological and pathological conditions. The aim of this dissertation thesis was to understand the effect of immune mediated inflammation on bone structural, mechanical and biological behaviour, using rheumatoid arthritis (RA) and fracture healing as a model. The first study of this thesis focused on the analysis of the effect of arthritis on bone biomechanical behavior. An animal model of arthritis, the SKG mice, was used and bone behavior was evaluated by mechanical three-point bending tests. Scanning electron microscopy (SEM) and multiphoton microscopy (MPM) were applied to study, respectively, bone structure and the collagen network organization in trabecular bone. Results from this study have shown that arthritic bones had poor biomechanical quality compared to control bones. MPM and SEM observations disclosed signs of impaired collagen organization and poor trabecular architecture. In this work we verified that chronic inflammation per se leads to impairment of bone biomechanics in terms of stiffness, ductility and ultimate strength. In the same model, we then proceed to study the effect of inflammation on collagen metabolism and organization. The evaluation of bone included mechanical tests, SEM, MPM and serum bone turnover markers, specifically procollagen type - amino-terminal peptide (P1NP) and carboxy-terminal crosslinked telopeptide of type I 6 collagen (CTX). Femoral bones of SKG mice revealed increased fragility expressed by deterioration of mechanical properties. In accordance, as observed by SEM, intertrabecular distance was increased and trabecular thickness decreased. MPM depicted a disorganized matrix and loose collagen structure compared to controls. Moreover, the collagen metabolism assessed in the serum was also highly increased in arthritic mice. In this work we found that the bone weakening effect of arthritis was due to high bone turnover and disorganized collagen type I matrix. Following these initial animal model data, the first to describe a direct effect of arthritis on collagen structure and bone biomechanics, we confirmed and further detailed these observations in human RA bone. Patients with RA submitted to hip replacement surgery were recruited. Trabecular bone microarchitecture was assessed by microcomputed tomography (microCT) and mechanical behavior by compression tests of a bone cylinder extracted from the femoral epiphysis. Bone cell activity was analyzed by studying gene expression in the bone microenvironment. Genes that code for proinflammatory cytokines were upregulated in RA patients, particularly IL-17, which plays an important role in stimulating osteoclastogenesis. RA bone microenvironment had a gene expression profile characterized by upregulated pro-osteoclastogenic cytokines and dickkopf homolog 1 (DKK1) and increased RANKL/OPG ratio. This was paralleled by raised expression of factors that promote osteoblastic activity, such as IGF-I, FGF2 and PDGF, but with low type I collagen expression. DKK1 is a negative regulator of the WNT/β- catenin signaling, which is a key pathway in the stimulation of osteoblast differentiation, meaning that its upregulation in RA patients is limiting the effects of pro-osteoblastic factors. Bone loss in a chronic inflammatory disease, such as RA, thus appears to result from enhanced bone resorption and impaired bone formation, which 7 constitutes a detrimental imbalance of bone remodeling and precipitates the rapid loss of bone mass. Indeed, differences were observed in gene expression between RA and primary osteoporosis (OP) bone in spite of the fact that these two patient groups had similar bone microarchitecture and biomechanical properties. These observations might indicate that the differences in gene expression reflect biologically specific mechanisms responsible for bone fragility in RA and that the inhibition of DKK1 can be a possible treatment strategy for tackling the effects of RA on bone. In the final work of this thesis, we have used the post-fracture inflammatory reaction as a model for characterizing the kinetics of inflammatory and bone remodeling related genes. Unlike the chronic inflammation seen in RA, fracture healing is a highly regulated and brief process. Patients submitted to hip replacement surgery after a low-energy hip fracture were enrolled in this study. Patients were grouped according to the time interval between fracture and surgery: bone collected within 3 days after fracture; between the 4th and 7th day; and one week after fracture. Inflammation and bone metabolism related genes were assessed at the fracture site. Our results indicate that the expression of inflammation related genes, especially IL-6, is highest at the very first days after fracture but from day 4 onwards there is a shift towards bone remodeling genes, suggesting that the inflammatory phase triggers bone healing. Sclerostin expression, an inhibitor of osteoblast differentiation, has an initial high expression level that is diminished after the reduction of inflammatory gene expression. We propose the existence of a two step process in bone healing, dependent on an initial inflammatory stimulus and a latter decrease in sclerostin-related effects, with a consequent proosteoblastic effect. Therefore, local promotion of these events might constitute a promising medical intervention to accelerate fracture healing. 8 The work herein discussed clearly shows that inflammation has a complex role in bone regulation. The identification of key regulators of this system will be crucial both for RA and fracture healing future management.O termo osteoimunologia foi usado pela primeira vez em 2000 para descrever a interacção entre células do sistema imunitário e do osso. Estes dois sistemas têm vários factores em comum, tais como citocinas, factores de transcrição e receptores. Consequentemente, a sua interacção ocorre, não só em condições fisiológicas, mas também patológicas. O objectivo desta dissertação de doutoramento é o de compreender o efeito da inflamação mediada pelo sistema imunitário no comportamento estrutural, mecânico e biológico do osso usando a artrite reumatóide e a regeneração de fracturas como modelos. O primeiro estudo desta tese foca a análise do efeito da artrite no comportamento biomecânico do osso. Foi utilizado um modelo animal de artrite, o ratinho SKG, para avaliar o comportamento biomecânico do osso por testes de flexão de 3 pontos. A microscopia electrónica de varrimento e a microscopia multifotão foram utilizadas, respectivamente, para o estudo da estrutura óssea e da organização da rede de colagénio no osso trabecular. Os resultados deste estudo mostraram que o osso de ratinhos com artrite apresenta pior qualidade biomecânica comparando com os controlos. Observações por microscopia multifotão e microscopia electrónica de varrimento demonstraram alteração da organização do colagénio e da estrutura trabecular. Neste trabalho verificámos que a inflamação crónica per se conduz à degradação das propriedades biomecânicas, particularmente da rigidez, ductilidade e resistência. No mesmo modelo animal, prosseguimos com o estudo do efeito da inflamação no metabolismo e organização do colagénio. A avaliação do osso incluiu ensaios mecânicos, microscopia electrónica de varrimento, microscopia multifotão e doseamento de marcadores 2 séricos de remodelação óssea, especificamente o péptido aminoterminal do procolagénio tipo I (P1NP) e o carboxi-terminal do telopéptido do colagénio tipo I (CTX). O osso dos ratinhos SKG revelou aumento da fragilidade óssea expresso pela deterioração das propriedades mecânicas. Em concordância, por microscopia electrónica de varrimento foi observado que a distância intertrabecular aumentou e a espessura trabecular reduziu e a microscopia multifotão demonstrou uma matriz desorganizada, com baixa densidade de colagénio. O metabolismo do colagénio foi ainda estudado no soro, encontrando-se aumentado nos ratinhos com artrite. Neste trabalho determinámos que o efeito de fragilidade óssea induzido pela artrite se deve à elevada remodelação óssea e desorganização da matriz de colagénio tipo I. Seguindo os dados iniciais obtidos em modelo animal, os primeiros a descrever o efeito directo da artrite na estrutura do colagénio e na biomecânica do osso, confirmámos e detalhámos estas observações em osso humano com artrite reumatóide. Foram recrutados doentes com AR submetidos a artroplastia da anca. A microarquitectura do osso trabecular foi avaliada por microtomografia computadorizada e o comportamento biomecânico por teste de compressão de um cilindro de osso trabecular extraído da epífise femoral. A actividade das células ósseas foi analisada através do estudo da expressão génica no microambiente ósseo. Observou-se um aumento da expressão dos genes que codificam citocinas pró-inflamatórias, particularmente a IL- 17, a qual desempenha um importante papel na estimulação da osteoclastogénese. O microambiente do osso com AR apresenta um perfil de expressão génico caracterizado pelo aumento das citocinas pró-osteoclastogénicas e de DKK1 e ainda do rácio de expressão RANKL/OPG. Paralelamente, verificou-se um aumento da expressão de factores indutores da actividade osteoblástica, tais como o IGF-I, FGF2 e PDGF, apesar da baixa expressão de colagénio tipo I. O DKK1 3 é um regulador negativo da via de sinalização WNT/β-catenina, sendo esta uma via chave na estimulação da diferenciação do osteoblasto. Assim, o aumento deste inibidor nos doentes com artrite reumatóide limita o efeito pró-osteoblastogénico do ambiente molecular do osso artrítico. A perda óssea associada às doenças crónicas inflamatórias, tal como a artrite reumatóide, parece, desta forma, resultar do aumento da reabsorção óssea e de uma diminuição na formação, conduzindo ao desequilíbrio da remodelação e precipitando a rápida perda de massa óssea. De facto, foram observadas diferenças na expressão génica entre o osso de doentes com artrite reumatóide e com osteoporose primária, apesar destes dois grupos de doentes terem microestrutura óssea e propriedades biomecânicas semelhantes. Estas observações parecem indicar que as diferenças encontradas a nível da expressão génica reflectem mecanismos biológicos específicos responsáveis pela fragilidade óssea na artrite reumatóide e a inibição do DKK1 poderá vir a ser uma possível estratégia terapêutica para diminuir os efeitos da artrite reumatóide sobre o osso. No último trabalho desta tese, utilizámos a reacção inflamatória pósfractura como modelo para caracterizar a cinética de expressão dos genes relacionados com a inflamação e remodelação óssea. Ao contrário da inflamação crónica, característica da artrite reumatóide, a regeneração de fracturas é um processo breve e cuidadosamente regulado. Foram incluídos neste estudo doentes submetidos a artroplastia da anca devido a fracturas de baixo impacto. Os doentes foram agrupados de acordo com o tempo decorrido entre a fractura e a cirurgia: osso colhido nos 3 dias após fractura, entre o 4º e o 7º dias, e uma semana após fractura. Um conjunto de genes relacionados com a inflamação e o metabolismo ósseo foram estudados no local de fractura. Os resultados obtidos indicam que a expressão dos genes relacionados com a inflamação, especialmente a 4 IL-6, estão aumentados nos primeiros dias após fractura; contudo, a partir do dia 4 verifica-se um desvio para os genes de remodelação óssea, sugerindo que a fase inflamatória activa a regeneração da fractura. A expressão da esclerostina, um inibidor da diferenciação do osteoblasto, está aumentada durante os primeiros dias após fractura e diminui após a redução da expressão dos genes pró-inflamatórios. Desta forma, propomos a existência de um processo em duas fases que conduz à regeneração de fracturas, dependente de um estímulo inicial inflamatório e uma diminuição subsequente dos efeitos relacionados com a esclerostina, com consequente estímulo osteoblástico. Assim, a indução local destes eventos poderá constituir uma intervenção promissora para acelerar a regeneração óssea após fractura. O trabalho aqui discutido claramente demonstra o complexo papel da inflamação na regulação óssea. A identificação de factores reguladores chave deste sistema será crucial para o futuro tratamento da artrite reumatóide e das fracturas

Differential tissue accumulation in the invasive Manila clam, Ruditapes philippinarum, under two environmentally relevant lanthanum concentrations

Among the environmental emerging concern rare earth elements, lanthanum (La) is one of the most common and reactive. Lanthanum is widely used in numerous modern technologies and applications, and its intense usage results in increasing discharges into the environment, with potentially deleterious consequences to earthlings. Therefore, we exposed the important food resource and powerful monitoring tool Manila clam to two environmentally relevant concentrations of La (0.3 µg L−1 and 0.9 µg L−1) for 6 days, through water, to assess the bioaccumulation pattern in the gills, digestive gland, and remaining body. The La bioaccumulation was measured after 1 (T1), 2 (T2), and 6 (T6) days of exposure. Lanthanum was bioaccumulated after 2 days, and the levels increased in all tissues in a dose-dependent manner. When exposed to 0.3 µg L−1, the enrichment factor pattern was gills > body > digestive gland. However, when exposed to 0.9 µg L−1, the pattern appears to change to gills > digestive gland > body. Tissue portioning appears to be linked with exposed concentration: In higher exposure levels, digestive gland seems to gain importance, probably associated with detoxification mechanisms. Here, we describe for the first time La bioaccumulation in these different tissues in a bivalve species. Future studies dealing with the bioaccumulation and availability of La should connect them with additional water parameters (such as temperature, pH, and major cations).Fundação para a Ciência e Tecnologia - FCTinfo:eu-repo/semantics/publishedVersio

Friend or foe?

Funding: This research was funded by Fundação para a Ciência e Tecnologia (FCT), PTDC/MECHEM/30315/2017.Multiple myeloma (MM) is one of the most prevalent hematological cancers worldwide, characterized by the clonal expansion of neoplastic plasma cells in the bone marrow (BM). A combination of factors is implicated in disease progression, including BM immune microenvironment changes. Increasing evidence suggests that the disruption of immunological processes responsible for myeloma control ultimately leads to the escape from immune surveillance and resistance to immune effector function, resulting in an active form of myeloma. In fact, one of the hallmarks of MM is the development of a permissive BM milieu that provides a growth advantage to the malignant cells. Consequently, a better understanding of how myeloma cells interact with the BM niche com-partments and disrupt the immune homeostasis is of utmost importance to develop more effective treatments. This review focuses on the most up-to-date knowledge regarding microenvironment-related mechanisms behind MM immune evasion and suppression, as well as promising molecules that are currently under pre-clinical tests targeting immune populations.publishersversionpublishe

Boosting immunity against multiple myeloma

Funding: This research was funded by Fundação para a Ciência e Tecnologia (FCT), PTDC/MECHEM/30315/2017 and UIDB/04443/2020Despite the improvement of patient’s outcome obtained by the current use of immunomod-ulatory drugs, proteasome inhibitors or anti-CD38 monoclonal antibodies, multiple myeloma (MM) remains an incurable disease. More recently, the testing in clinical trials of novel drugs such as anti-BCMA CAR-T cells, antibody–drug conjugates or bispecific antibodies broadened the possibility of improving patients’ survival. However, thus far, these treatment strategies have not been able to steadily eliminate all malignant cells, and the aim has been to induce a long-term complete response with minimal residual disease (MRD)-negative status. In this sense, approaches that target not only myeloma cells but also the surrounding microenvironment are promising strategies to achieve a sustained MRD negativity with prolonged survival. This review provides an overview of current and future strategies used for immunomodulation of MM focusing on the impact on bone marrow (BM) immunome.publishersversionpublishe

Warming enhances lanthanum accumulation and toxicity promoting cellular damage in glass eels (Anguilla anguilla)

Cumulative and continuing human emissions of greenhouse gases to the atmosphere are causing ocean warming. Rising temperature is a major threat to aquatic organisms and may affect physiological responses, such as acid-base balance, often compromising species fitness and survival. It is also expected that warming may influence the availability and toxicological effects of pollutants, including Rare Earth Elements. These are contaminants of environmental emerging concern with great economic interest. This group comprises yttrium, scandium and lanthanides, being Lanthanum (La) one of the most common. The European eel (Anguilla anguilla) is critically endangered and constitutes a delicacy in South East Asia and Europe, being subject to an increasing demand on a global scale. Considering the vulnerability of early life stages to contaminants, we exposed glass eels to 1.5 μg L-1 of La for five days, plus five days of depuration, under a present-day temperature and warming scenarios (△T = +4 °C). The aim of this study was to assess the bioaccumulation, elimination and specific biochemical enzymatic endpoints in glass eels (Anguilla anguilla) tissues, under warming and La. Overall, our results showed that the accumulation and toxicity of La were enhanced with increasing temperature. The accumulation was higher in the viscera, followed by the head, and ultimately the body. Elimination was less effective under warming. Exposure to La did not impact acetylcholinesterase activity. Moreover, lipid peroxidation peaked after five days under the combined exposure of La and warming. The expression of heat shock proteins was majorly suppressed in glass eels exposed to La, at both tested temperatures. This result suggests that, when exposed to La, glass eels were unable to efficiently prevent cellular damage, with a particularly dramatic setup in a near-future scenario. Further studies are needed towards a better understanding of the effects of lanthanum in a changing world.info:eu-repo/semantics/publishedVersio

Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Rheumatoid Arthritis

Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14(bright) CD16-monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6.Peer reviewe

Low Serum Levels of DKK2 Predict Incident Low-Impact Fracture in Older Women

There are currently no robust noninvasive markers of fragility fractures. Secreted frizzled related protein-1 (sFRP-1), dickkopf-related protein 1 (DKK1) and DKK2, and sclerostin (SOST) inhibit Wnt signaling and interfere with osteoblast-mediated bone formation. We evaluated associations of serum levels of sFRP-1, DKK1, DKK2, and SOST with incident low-impact fracture and BMD in 828 women aged ≥65 years from EpiDoC, a longitudinal population-based cohort. A structured questionnaire during a baseline clinical appointment assessed prevalent fragility fractures and clinical risk factors (CRFs) for fracture. Blood was collected to measure serum levels of bone turnover markers and Wnt regulators. Lumbar spine and hip BMD were determined by DXA scanning. Follow-up assessment was performed through a phone interview; incident fragility fracture was defined by any new self-reported low-impact fracture. Multivariate Cox proportional hazard models were used to analyze fracture risk adjusted for CRFs and BMD. During a mean follow-up of 2.3 ± 1.0 years, 62 low-impact fractures were sustained in 58 women. A low serum DKK2 level (per 1 SD decrease) was associated with a 1.5-fold increase in fracture risk independently of BMD and CRFs. Women in the two lowest DKK2 quartiles had a fracture incidence rate of 32 per 1000 person-years, whereas women in the two highest quartiles had 14 fragility fractures per 1000 person-years. A high serum sFRP1 level was associated with a 1.6-fold increase in fracture risk adjusted for CRFs, but not independently of BMD. Serum levels of SOST (r = 0.191; p = 0.0025) and DKK1(r = -0.1725; p = 0.011) were correlated with hip BMD, but not with incident fragility fracture. These results indicate that serum DKK2 and sFRP1 may predict low-impact fracture. The low number of incident fractures recorded is a limitation and serum levels of Wnt regulators should be further studied in other populations as potential noninvasive markers of fragility fractures. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.publishersversionpublishe

Ankylosing Spondylitis Patients Have Impaired Osteoclast Gene Expression in Circulating Osteoclast Precursors

Introduction: Ankylosing spondylitis (AS) is typically characterized by focal bone over-growth and also by systemic bone loss. We hypothesize that the increased osteoproliferation found in AS might be partially due to reduced ability of osteoclast precursors (OCPs) to differentiate into osteoclasts (OCs). Therefore, our aim was to characterize bone remodeling and pro-osteoclastogenesis inflammatory environment, monocytes' phenotype, and in vitro osteoclast differentiation in AS patients. Methods: Patients with active AS without any ongoing therapy and age-and gender matched healthy donors were recruited. Receptor activator of nuclear factor-K13 (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations were assessed. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and quantitative reverse transcription real-time PCR for OC-specific genes were performed. Results: Pro-and anti-inflammatory cytokine serum levels were higher in AS patients than in controls. RANKL neutrophil expression was higher in AS patients when compared to healthy donors, but CD51/CD61 expression was lower in the classical monocyte subpopulation. Concerning osteoclastogenesis, we found no differences in the in vitro osteoclast differentiating potential of these cells when compared to healthy donors. However, we observed low expression of CSF1R, RANK, and NFATc1 in AS OCPs. Conclusion: Despite the high levels of pro-inflammatory cytokines present in AS patients, no differences in the number of OC or resorbed area were found between AS patients and healthy donors. Moreover, we observed that OCPs have low OC specific gene expression. These findings support our hypothesis of an impaired response of OCPs to pro-osteoclastogenic stimuli in vivo in AS patients.Peer reviewe

Gadolinium ecotoxicity is enhanced in a warmer and acidified changing ocean as shown by the surf clam Spisula solida through a multibiomarker approach

Funding Information: This work was supported by Fundação para a Ciência e Tecnologia (FCT) , through the project Climatoxeel ( PTDC/AAG-GLO/3795/2014 ), by the Junior Researcher contract ( CEECIND/03517/2017 ), both awarded to Tiago F. Grilo, and the strategic project UIDB/04292/2020 granted to MARE and through project LA/P/0069/2020 granted to the Associate Laboratory ARNET . The work was also supported by the European Union's operation program Mar 2020 through the research project CEIC ( MAR-01.04.02-FEAMP-0012 ) awarded to Joana Raimundo. The Applied Molecular Biosciences Unit UCIBIO was financed by national funds from FCT ( UIDP/04378/2020 ). This work was also supported by the European Union through the grant ERC-2016-COG-725034 -ecotox awarded to Inês João Ferreira. Cátia Figueiredo acknowledges the FCT-PhD grant SFRH/BD/130023/2017 and the Early Career Research Grant awarded by National Geographic Society. Publisher Copyright: © 2022 The Author(s)Humans have exhaustively combusted fossil fuels, and released pollutants into the environment, at continuously faster rates resulting in global average temperature increase and seawater pH decrease. Climate change is forecasted to exacerbate the effects of pollutants such as the emergent rare earth elements. Therefore, the objective of this study was to assess the combined effects of rising temperature (Δ = + 4 °C) and decreasing pH (Δ = − 0.4 pH units) on the bioaccumulation and elimination of gadolinium (Gd) in the bioindicator bivalve species Spisula solida (Surf clam). We exposed surf clams to 10 µg L−1 of GdCl3 for seven days, under warming, acidification, and their combination, followed by a depuration phase lasting for another 7 days and investigated the Gd bioaccumulation and oxidative stress-related responses after 1, 3 and 7 days of exposure and the elimination phase. Gadolinium accumulated after just one day with values reaching the highest after 7 days. Gadolinium was not eliminated after 7 days, and elimination is further hampered under climate change scenarios. Warming and acidification, and their interaction did not significantly impact Gd concentration. However, there was a significant interaction on clam's biochemical response. The augmented total antioxidant capacity and lipid peroxidation values show that the significant impacts of Gd on the oxidative stress response are enhanced under warming while the increased superoxide dismutase and catalase values demonstrate the combined impact of Gd, warming & acidification. Ultimately, lipid damage was greater in clams exposed to warming & Gd, which emphasizes the enhanced toxic effects of Gd in a changing ocean.publishersversionpublishe