Catalpol protects against 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a well‐known environmental contaminant that produces a wide variety of adverse effects in humans. Catalpol, a major bioactive compound enriched in the dried root of Rehmannia glutinosa, is a major iridoid glycoside that alleviates bone loss. However, the detailed mechanisms underlying the effects of catalpol remain unclear. The present study evaluated the effects of catalpol on TCDD‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells. Catalpol inhibited TCDD‐induced reduction in cell viability and increases in apoptosis and autophagic activity in osteoblastic MC3T3‐E1 cells. Additionally, pretreatment with catalpol significantly decreased the nitric oxide and nitrite levels compared with a control in TCDD‐treated cells and significantly inhibited TCDD‐induced increases in the levels of cytochrome P450 1A1 and extracellular signal‐regulated kinase. Pretreatment with catalpol also effectively restored the expression of superoxide dismutase and extracellular signal‐regulated kinase 1 and significantly enhanced the expression of glutathione peroxidase 4 and osteoblast differentiation markers, including alkaline phosphatase and osterix. Taken together, these findings demonstrate that catalpol has preventive effects against TCDD‐induced damage in MC3T3‐E1 osteoblastic cells.     

Actein alleviates 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐mediated cellular dysfunction in osteoblastic MC3T3‐E1 cells

The environmental pollutant 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is known to affect bone metabolism. We evaluated the protective effects of the triterpene glycoside actein from the herb black cohosh against TCDD‐induced toxicity in MC3T3‐E1 osteoblastic cells. We found that TCDD significantly reduced cell viability and increased apoptosis and autophagy in MC3T3‐E1 osteoblastic cells (P < .05). In addition, TCDD treatment resulted in a significant increase in intracellular calcium concentration, mitochondrial membrane potential collapse, reactive oxygen species (ROS) production, and cardiolipin peroxidation, whereas pretreatment with actein significantly mitigated these effects (P < .05). The effects of TCDD on extracellular signal‐related kinase (ERK), aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and cytochrome P450 1A1 levels in MC3T3‐E1 cells were significantly inhibited by actein. The levels of superoxide dismutase, ERK1, and nuclear factor kappa B mRNA were also effectively restored by pretreatment with actein. Furthermore, actein treatment resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblastic differentiation (ALP, type I collagen, osteoprotegerin, bone sialoprotein, and osterix). This study demonstrates the underlying molecular mechanisms of cytoprotection exerted by actein against TCDD‐induced oxidative stress and osteoblast damage.     

Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3‐E1 cells against antimycin A‐induced cytotoxicity

Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)‐induced toxicity in osteoblastic MC3T3‐E1 cells. Exposure of MC3T3‐E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca²⁺]_i) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A‐induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca²⁺]_i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3‐kinase), Akt (protein kinase B) and CREB (cAMP‐response element‐binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. Copyright © 2011 John Wiley & Sons, Ltd.     

Cytoprotective effects of xanthohumol against methylglyoxal‐induced cytotoxicity in MC3T3‐E1 osteoblastic cells

Methylglyoxal (MG) has been suggested to be a major source of intracellular reactive carbonyl compounds, and has been implicated in increasing the levels of advanced glycation end products in age‐related diseases. Xanthohumol is a prenylated flavonoid found in hops (Humulus lupulus) and beer. In the present study, we investigated the effects of xanthohumol on MG‐induced cytotoxicity in osteoblastic MC3T3‐E1 cells. Xanthohumol attenuated MG‐induced cytotoxicity, as evidenced by improved cell viability, and prevented MG‐induced MG‐protein adducts, inflammatory cytokines, reactive oxygen species and mitochondrial superoxide production. In addition, xanthohumol increased glyoxalase I activity, glutathione, heme oxygenase‐1 and nuclear factor erythroid 2‐related factor 2 levels in the presence of MG. Pretreatment with xanthohumol before MG exposure reduced MG‐induced mitochondrial dysfunction. Furthermore, xanthohumol treatment resulted in a significant reduction in the levels of endoplasmic reticulum stress and autophagy induced by MG. Notably, the autophagy‐reducing effect of xanthohumol was abolished after the addition of Ex527, a selective inhibitor of sirtuin 1, suggesting that xanthohumol is an effective sirtuin 1 activator for reducing autophagy. Taken together, our findings suggest xanthohumol as a promising new strategy for preventing diabetic osteopathy.     

Inhibitory effect of apocynin on methylglyoxal‐mediated glycation in osteoblastic MC3T3‐E1 cells

Methylglyoxal (MG), a highly reactive metabolite of hyperglycemia, can enhance protein glycation, oxidative stress or inflammation. The present study investigated the effects of apocynin on the mechanisms associated with MG toxicity in osteoblastic MC3T3‐E1 cells. Pretreatment of MC3T3‐E1 cells with apocynin prevented the MG‐induced protein glycation and formation of intracellular reactive oxygen species and mitochondrial superoxide in MC3T3‐E1 cells. In addition, apocynin increased glutathione levels and restored the activity of glyoxalase I inhibited by MG. These findings suggest that apocynin provide a protective action against MG‐induced cell damage by reducing oxidative stress and by increasing the MG detoxification system. Apocynin treatment decreased the levels of proinflammatory cytokines such as tumor necrosis factor‐α and interleukin‐6 induced by MG. Additionally, the nitric oxide level reduced by MG was significantly increased by apocynin. These findings indicate that apocynin might exert its therapeutic effects via upregulation of glyoxalase system and antioxidant activity. Taken together, apocynin may prove to be an effective treatment for diabetic osteopathy. Copyright © 2014 John Wiley & Sons, Ltd.     

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD)‐Induced Activation of Mitogen‐Activated Protein Kinase Signaling Pathway in Jurkat T Cells

Abstract: The present study was performed to examine mitogen‐activated protein kinase associated pathways in mediation of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD)‐induced cell apoptosis in cultured Jurkat T cells. TCDD significantly decreased cell viability in a concentration‐dependent manner (P<0.05 at 10–300 nM). TCDD (10 nM) also time‐dependently decreased cell viability (P<0.05 at 12–48 hr). c‐Jun NH₂‐terminal kinase was significantly phosphorylated with TCDD treatment in a time dependent manner. p38 Mitogen‐activated protein kinase was not significantly changed with TCDD treatment. Extracellular signal‐regulated protein kinase was significantly phosphorylated with TCDD treatment for 8 hr and gradually returned to baseline. TCDD induced up‐regulation of ASK1 and C‐Jun, which are up‐ and down‐stream of JNK, respectively, and up‐regulation of cytosolic cytochrome c and caspase‐3. These results demonstrate that MAPK signaling pathways including JNK and ERK 1/2, are activated with the treatment of TCDD in Jurkat T cells, which suggest that MAPK pathways may be involved in TCDD‐induced cell death.     

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin exposure influence the expression of glutamate transporter GLT‐1 in C6 glioma cells via the Ca2+/protein kinase C pathway

The widespread environmental contaminant, 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), is considered one of the most toxic dioxin‐like compounds. Although epidemiological studies have shown that TCDD exposure is linked to some neurological and neurophysiological disorders, the underlying mechanism of TCDD‐mediated neurotoxicity has remained unclear. Astrocytes are the most abundant cells in the nervous systems, and are recognized as the important mediators of normal brain functions as well as neurological, neurodevelopmental and neurodegenerative brain diseases. In this study, we investigated the role of TCDD in regulating the expression of glutamate transporter GLT‐1 in astrocytes. TCDD, at concentrations of 0.1–100 nm , had no significantly harmful effect on the viability of C6 glioma cells. However, the expression of GLT‐1 in C6 glioma cells was downregulated in a dose‐ and time‐dependent manner. TCDD also caused activation of protein kinase C (PKC), as TCDD induced translocation of the PKC from the cytoplasm or perinuclear to the membrane. The translocation of PKC was inhibited by one Ca²⁺ blocker, nifedipine, suggesting that the effects are triggered by the initial elevated intracellular concentration of free Ca²⁺. Finally, we showed that inhibition of the PKC activity reverses the TCDD‐triggered reduction of GLT‐1. In summary, our results suggested that TCDD exposure could downregulate the expression of GLT‐1 in C6 via Ca²⁺/PKC pathway. The downregulation of GLT‐1 might participate in TCDD‐mediated neurotoxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Deoxyactein stimulates osteoblast function and inhibits bone‐resorbing mediators in MC3T3‐E1 cells

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In order to improve the treatment of osteoporosis, identification of anabolic agents with minimal side effects is highly desirable. Cimicifuga racemosa has a long and diverse history of medicinal use and deoxyactein isolated from this species is one of the major constituents. In the present study, the effect of deoxyactein on the function of osteoblastic MC3T3‐E1 cells was studied. Deoxyactein caused a significant elevation of cell growth, alkaline phosphatase activity, collagen content, and mineralization in the cells (P < 0.05). Moreover, deoxyactein significantly (P < 0.05) decreased the production of reactive oxygen species (ROS) and osteoclast differentiation‐inducing factors such as TNF‐α, IL‐6 and receptor activator of nuclear factor‐κB ligand in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as an ROS generator. These results demonstrate that deoxyactein may have positive effects on skeletal structure. Copyright © 2011 John Wiley & Sons, Ltd.     

Different mechanisms of action of 2, 2’, 4, 4’‐tetrabromodiphenyl ether (BDE‐47) and its metabolites (5‐OH‐BDE‐47 and 6‐OH‐BDE‐47) on cell proliferation in OVCAR‐3 ovarian cancer cells and MCF‐7 breast cancer cells

Data concerning the possible action of polybrominated diphenyl ethers (PBDEs) in hormone‐dependent cancer are scarce. Some data showed that PBDEs may directly affect breast cancer cells formation and only one research showed increased proliferation of the OVCAR‐3 cells, but the results are ambiguous and the mechanisms are not clear. There is growing evidence that not only parent compounds but also its metabolites may be involved in cancer development. The present study was, therefore, designed to determine the effect of BDE‐47 and its metabolites (2.5 to 50 ng ml^–1) on proliferation (BrdU), cell‐cycle genes (real‐time PCR) and protein expression (Western blot), protein expression of oestrogen receptors (α β), extracellular signal‐regulated kinases 1 and 2 (ERK1/2) and protein kinase Cα (PKCα) in OVCAR‐3 ovarian and MCF‐7 breast cancer cells. In OVCAR‐3 cells, the parent compound stimulated cell proliferation by activating CDK1, CDK7, E2F1 and E2F2. Independent of time of exposure, BDE‐47 had no effect on ERα and ERβ protein expression and ERK1/2 and PKCα phosphorylation. Metabolites had no effect on cell proliferation but increased both ERs protein expression and ERK1/2 and PKCα phosphorylation. In MCF‐7 cells, the parent compound displayed no effect on cell proliferation but decreased ERα and increased ERβ protein expression with concomitant induction of PKCα phosphorylation. Both metabolites increased MCF‐7 cell proliferation, ERK1/2 and PKCα phosphorylation and decreased ERα and ERβ protein expression.We suggest that studies concerning PBDEs with fewer bromine atoms should be continued to understand environmental links to different hormone‐dependent cancers. Copyright © 2016 John Wiley & Sons, Ltd.     

14‐3‐3 inhibits insulin‐like growth factor‐I‐induced proliferation of cardiac fibroblasts via a phosphatidylinositol 3‐kinase‐dependent pathway

1. Insulin‐like growth factor (IGF)‐I plays an important role in the pathogenesis of heart disease and has been shown to strongly induce the proliferation of cardiac fibroblasts (CFs). It remains unknown whether 14‐3‐3 proteins, which are associated the regulation of signal transduction, affect IGF‐I‐induced CF proliferation. 2. In the present study, we investigated the effects of 14‐3‐3 proteins on CF proliferation in response to IGF‐I. Proliferation of CFs was determined by cell counting and a bromodeoxyuridine incorporation assay. Phosphorylation of signalling molecules was evaluated by western blottling. Activity of nuclear factor of activated T cells (NFAT) was examined using a dual luciferase reporter gene assay and immunofluorescence. 3. It was found that adenovirus‐mediated transfection of YFP‐R18 peptide (AdR18), a known inhibitor of 14‐3‐3, significantly enhanced IGF‐I‐induced CF proliferation. This potentiation arose from an increase in phosphorylation of phosphatidylinositol 3‐kinase (PI3‐K) and AKT (protein kinase B), inactivation of glycogen synthesis kinase (GSK) 3β and increased NFAT activity. 4. Collectively, the results of the present study suggest that 14‐3‐3 proteins inhibit IGF‐I‐induced CF proliferation via a PI3‐K‐dependent NFAT signalling pathway. This finding may contribute to our understanding of the function of 14‐3‐3 proteins in the heart.     

HEK293 cells exposed to microcystin‐LR show reduced protein phosphatase 2A activity and more stable cytoskeletal structure when overexpressing α4 protein

Microcystin‐LR (MC‐LR) is one of the most toxic members of microcystins released by freshwater cyanobacterial. The major mechanism of MC‐LR toxicity has been attributed to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A). In our prior research, α4 protein, a regulator of PP2A, was found not only crucial for PP2A regulation but also for the overall response of HEK 293 cells encountering MC‐LR. To explore the role of α4 in MC‐LR toxicity via PP2A regulation, here, HEK 293 cells overexpressing α4 protein were exposed to MC‐LR and PP2A, cytoskeletal organization, and cytoskeleton‐related proteins were investigated. The results showed that PP2A activity decreased and PP2A/C subunit expression and phosphorylation at Tyr307 increased significantly in the group exposed to high MC‐LR. Vimentin IF became concentrated and formed perinuclear bundles. However, the assembly of actin filament and microtubules remained unchanged and the expression and phosphorylation of the cytoskeleton‐related proteins HSP27 and VASP did not increase significantly. Some of these results differ from those of our previous study in which normal HEK293 cells were exposed to MC‐LR. Our results indicate that elevated α4 expression confers some resistance to MC‐LR‐induced cytoskeletal change These new findings provide helpful insights into the mechanism of MC‐LR toxicity and the role of α4 in regulating PP2A function. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 255–264, 2017.     

A genomic characterization of the influence of silver nanoparticles on bone differentiation in MC3T3‐E1 cells

Silver nanoparticles (AgNPs) have been widely used in a variety of biomedical applications. Previous studies demonstrated that AgNPs significantly enhanced bone cell mineralization and differentiation in MC3T3–1 cells, a model in vitro system, when compared to several other NPs. This increased bone deposition was evaluated by phenotypic measurements and assessment of the expression of miRNAs associated with regulation of bone morphogenic proteins. In the present study, we used RNA‐seq technology, a more direct measurement of gene expression, to investigate further the mechanisms of bone differentiation induced by AgNP treatment. Key factors associated with the osteoclast pathway were significantly increased in response to AgNP exposure including Bmp4, Bmp6 and Fosl1. In addition, genes of metabolism and toxicity pathways were significantly regulated as well. Although this study suggests the potential for AgNPs to influence bone morphogenesis in injury or disease applications, further investigation into the efficacy and safety of AgNPs in bone regeneration is warranted.     

Role of protein kinase C in bradykinin-induced prostaglandin formation in osteoblasts

Bradykinin (1 μM 5 min) induced translocation of protein kinase C (PKC) to the plasma membrane fraction in osteoblastic MC3T3-E1 cells. Bradykinin also enhanced the binding of phorbol 12,13-dibutyrate (PDBu) to intact cells, a measure of PKC activation. Addition of bradykinin (1 μM) to cells preincubated with [³H]PDBu (10 nM, 20 min) caused an increase in specific PDBu binding that was maximal after 5–10 min. The bradykinin-induced enhancement of PDBu binding was seen at 1 nM and was maximal at 10 nM. The bradykinin B1 receptor agonist des-Arg⁹-bradykinin (1 μM) did not enhance specific PDBu binding to intact MC3T3-E1 cells. PDBu at and above 3 nM stimulated the formation of prostaglandin E2 (PGE₂) in MC3T3-EI cells. This stimulatory effect was seen after 15–20 min incubation. The Ca²⁺ ionophore A23187 at and above 1 μM induced a rapid (within seconds) burst of PGE₂ formation in MC3T3-E1 cells. The effect of PDBu and A23187 on PGE₂ formation was synergistic. The PKC inhibitor staurosporine (200 nM) inhibited basal as well as bradykinin-induced prostaglandin-formation in MC3T3-E1 cells. In conclusion: bradykinin enhances PKC activation in osteoblastic MC3T3-E1 cells. This kinase activation may be involved in bradykinin-induced prostaglandin formation.     

Effects of bergenin on methylglyoxal‐induced damage in osteoblastic MC3T3‐E1 cells

Bergenin is the main chemical constituent of plants in the genus Bergenia, which are used in traditional medicines. Methylglyoxal (MG), a highly reactive dicarbonyl compound, is the major precursor for forming advanced glycation end products (AGEs). Pretreating MC3T3‐E1 cells with bergenin prevented MG‐induced protein adduct formation. Bergenin inhibited the MG‐induced soluble receptor for AGE (sRAGE), interleukin, reactive oxygen species and mitochondrial superoxide production. Additionally bergenin increased glyoxalase I activity, glutathione, heme oxygenase‐1 and nuclear factor erythroid 2‐related factor 2 levels in the presence of MG. Pretreatment with bergenin before MG exposure reduced MG‐induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation, loss of adenosine triphosphate and reduced adenosine monophosphate‐activated protein kinase. These results demonstrate that bergenin may prevent the development of diabetic osteopathy.     

2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin induces premature senescence of astrocytes via WNT/β‐catenin signaling and ROS production

2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental contaminant that could exert significant neurotoxicity in the human nervous system. Nevertheless, the molecular mechanism underlying TCDD‐mediated neurotoxicity has not been clarified clearly. Herein, we investigated the potential role of TCDD in facilitating premature senescence in astrocytes and the underlying molecular mechanisms. Using the senescence‐associated β‐galactosidase (SA‐β‐Gal) assay, we demonstrated that TCDD exposure triggered significant premature senescence of astrocyte cells, which was accompanied by a marked activation of the Wingless and int (WNT)/β‐catenin signaling pathway. In addition, TCDD altered the expression of senescence marker proteins, such as p16, p21 and GFAP, which together have been reported to be upregulated in aging astrocytes, in both dose‐ and time‐dependent manners. Further, TCDD led to cell‐cycle arrest, F‐actin reorganization and the accumulation of cellular reactive oxygen species (ROS). Moreover, the ROS scavenger N‐acetylcysteine (NAC) markedly attenuated TCDD‐induced ROS production, cellular oxidative damage and astrocyte senescence. Notably, the application of XAV939, an inhibitor of WNT/β‐catenin signaling pathway, ameliorated the effect of TCDD on cellular β‐catenin level, ROS production, cellular oxidative damage and premature senescence in astrocytes. In summary, our findings indicated that TCDD might induce astrocyte senescence via WNT/β‐catenin and ROS‐dependent mechanisms. Copyright © 2014 John Wiley & Sons, Ltd.     

T‐bet interferes with PD‐1/PD‐L1‐mediated suppression of CD4+ T cell inflammation and survival in Crohn's disease

Pathogenic inflammation mediated by overactive type 1 helper T cell (Th1) responses could exacerbate and perpetuate Crohn's disease. Programmed death (PD)‐1 and its ligand PD‐L1 pathway could be upregulated to suppress inflammation. We wondered why this pathway is ineffective at suppressing pathogenic Th1 inflammation in Crohn's disease patients. Here, we found that overexpression of T‐bet via transfection significantly reduced the expression of PD‐1. PD‐L1 was capable of suppression proinflammatory CD4⁺ T cells, but T‐bet transfection significantly reduced the susceptibility of CD4⁺ T cells toward PD‐L1‐mediated suppression, evidenced by the observations that at low PD‐L1 concentration T‐bet transfected and mock transfected CD4⁺ T cells presented comparable IL‐2 production, but at high PD‐L1 concentration, T‐bet transfected CD4⁺ T cells presented significantly higher IL‐2 than mock transfected CD4⁺ T cells. PD‐L1 could significantly reduce the survival of CD4⁺ T cells from Crohn's disease patients, but interestingly, in the absence of PD‐L1, the survival was better in mock transfected CD4⁺ T cells, while in the presence of PD‐L1, the survival was better in T‐bet transfected CD4⁺ T cells. Crohn's disease patients with greater severity presented higher T‐bet expression and lower PD‐1 expression in CD4⁺ T cells, demonstrating an association between T‐bet expression and disease progression. We also discovered that stimulation with bacterial antigens could upregulate the expression of T‐bet. Together, this study demonstrated that T‐bet overexpression could interfere with PD‐1/PD‐L1‐mediated suppression of CD4⁺ T cell inflammation and survival, and potentially contributed to the development and persistence of Crohn's disease.     

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) Increases Bilirubin Formation but Hampers Quantitative Hepatic Conversion of Biliverdin to Bilirubin in Rats with Wild‐Type AH Receptor

In haem degradation, haem oxygenase‐1 (HO‐1) first cleaves haem to biliverdin, which is reduced to bilirubin by biliverdin IXα reductase (BVR‐A). The environmental pollutant 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) causes hepatic accumulation of biliverdin in moderately TCDD‐resistant line B (Kuopio) rats. Using line B and two TCDD‐sensitive rat strains, the present study set out to probe the dose–response and biochemical mechanisms of this accumulation. At 28 days after exposure to 3–300 μg/kg TCDD in line B rats, already the lowest dose of TCDD tested, 3 μg/kg, affected serum bilirubin conjugates, and after doses ≥100 μg/kg, the liver content of bilirubin, biliverdin and their conjugates (collectively ‘bile pigments’) as well as HO‐1 was elevated. BVR‐A activity and serum bile acids were increased only by the doses of 100 and 300 μg/kg TCDD, respectively. Biliverdin conjugates correlated best with biliverdin suggesting it to be their immediate precursor. TCDD (100 μg/kg, 10 days) increased hepatic bilirubin and biliverdin levels also in TCDD‐sensitive Long‐Evans (Turku/AB; L‐E) rats. Hepatic bilirubin and bile acids, but not biliverdin, were increased in feed‐restricted L‐E control rats. In TCDD‐sensitive line C (Kuopio) rats, 10 μg/kg of TCDD increased the body‐weight‐normalized biliary excretion of bilirubin. Altogether, the results suggest that at acutely toxic doses, TCDD induces the formation of bilirubin in rats. However, concurrently, TCDD seems to hamper the quantitative conversion of biliverdin to bilirubin in line B and L‐E rats' liver. Biliverdin conjugates are most likely formed as secondary products of biliverdin.