SPOTLIGHT pubs.acs.org/crt
TRANSFERRIN DELIVERS TOXIC PLUTONIUM Plutonium, a radiotoxic product of nuclear fission, is an important environmental contaminant at sites of nuclear accidents and prior nuclear testing. Pu is concentrated in the liver and skeleton, where it can remain for decades, causing considerable toxic damage; however, the means by which Pu gains entry into cells is currently uncertain. Pu(IV), the primary oxidation state of Pu under physiologic conditions, binds to the iron transport and storage proteins transferrin (Tf) and ferritin. Tf is a bilobar serum protein which binds one Fe3+ ion at its C-terminal lobe and one at its N-terminal lobe. Both lobes of Fe2Tf adopt a closed conformation, and this form of the protein binds to the Tf receptor (TR) for uptake into cells. Pu4+ binds to Tf with high affinity, but Pu2Tf does not undergo TR-mediated cellular uptake. Now, Jensen et al. [(2011) Nat.Chem. Biol., 7, 560], demonstrate Tf/TRdependent internalization of Pu4+ in the presence of Fe3+. Jensen et al. constructed Tf complexes containing Pu4+ in one lobe and Fe3+ in the other lobe as well as complexes containing two identical ions. Using a direct binding assay, they demonstrated that the affinity of PuCFeNTf for the TR was nearly as high as that of Fe2Tf. In contrast FeCPuNTf and Pu2Tf exhibited a much lower TR binding affinity. Small-angle X-ray scattering analysis revealed similar solution structures for PuCFeNTf and Fe2Tf, with both lobes closed in each case. In contrast, the structures of FeCPuNTf and Pu2Tf suggested the presence of one open lobe, most likely the Pu4+-containing N-lobe. These results suggest that the C-lobe can accommodate the larger Pu4+ ion and adopt the closed conformation, whereas the N-lobe cannot. Incubation of PC12 rat adrenal cells with each form of Tf resulted in Pu4+ uptake in the case of PuCFeNTf but not FeCPuNTf. Pu4+ uptake in the presence of PuCFeNTf was temperature-dependent and inhibited in the presence of Fe2Tf or chloroquine. These observations support the hypothesis that Pu4+ uptake in PuCFeNTf-exposed cells was TR-mediated. Under normal physiological conditions, approximately 45% of serum Tf is in the FeNTf form, which could bind Pu4+ to form PuCFeNTf. Thus, this mechanism may mediate Pu4+ entry into cells in vivo, and the possibility exists that other toxic metals may also gain cellular access via binding to Tf’s C-lobe in the presence of Fe3+. Carol A. Rouzer
’ DRUG METABOLISM FROM MICE TO MAN
Reprinted with permission from Chen et al. (2011) Proc. Natl. Acad. Sci. U.S.A., 108, 11842. Copyright 2011, Chen et al. Animal models have been a mainstay for the preclinical evaluation of new drug metabolism and toxicity. However, species differences in metabolic enzymes may result in the failure to detect toxic human metabolites, leading to adverse outcomes in the costly clinical phase of drug development. A promising solution to this problem is the development of humanized mice, which, through genetic manipulation or allogeneic transplantation, express human forms of key metabolic enzymes. Now, Chen et al. [(2011) Proc. Natl. Acad. Sci. U.S.A., 108, 11842] describe human ectopic artificial livers (HEALs) as an efficient way to generate humanized mice for metabolic studies. Chen et al. constructed HEALs by cocultivating primary human hepatocytes with J2-3T3 mouse fibroblasts for one week and then encapsulating the cells together with human liver endothelial cells (TMNK-1) in a polyethylene glycol diacrylate (PEG-DA) polymer scaffold. The scaffold included the fibronectin-derived peptide RGDS. The resulting three-dimensional cultures exhibited a high level of cellular stability and hepatocyte function for periods of over three weeks. When compared to previously characterized two-dimensional hepatocyte fibroblast cocultures, HEALs expressed similar or r 2011 American Chemical Society
higher levels of 68 out of 82 genes that are relevant to drug metabolism. Furthermore, exposure of HEALs to omeprazole or rifampin led to selective increases in the activity of cytochromes P450 1A2 and 3A4, respectively, indicating an ability to respond to inducers of P450 gene expression. HEALs also responded to P450-inhibiting compounds in the expected fashion. Intraperitoneal implantation of HEALs into mice led to efficient engraftment in less than one week, and human hepatic proteins could be detected in the mice for periods of at least several weeks. The HEAL-humanized mice metabolized coumarin and debrisoquine to the human-specific metabolites, 7-hydroxycoumarin and 4-hydroxydebrisoquine, respectively. HEALs explanted from rifampin-treated mice exhibited a selective 5-fold induction of P450 3A4 activity. Furthermore, rifampin-treated HEAL-humanized mice developed human hepatotoxicity upon administration of a therapeutic dose of acetaminophen as a result of increased P450 3A4-mediated conversion of acetaminophen to its reactive metabolite N-acetyl-p-benzoquinone. These results confirm that HEAL-humanized mice demonstrate species-specific human patterns of drug metabolism, including drug drug interactions. HEALs allow a rapid and efficient generation of the humanized mouse model, and HEAL implantation does not require prior liver injury or immunocompromised animals as in the case of other forms of allogeneic transplantation. This model provides a promising new approach to the preclinical evaluation of drug metabolism and toxicity as well as a means to explore human liver disease processes and therapies. Carol A. Rouzer Published: October 17, 2011 1599
dx.doi.org/10.1021/tx2003883 | Chem. Res. Toxicol. 2011, 24, 1599–1600
Chemical Research in Toxicology
’ LIPIDOMICS REVEALS MECHANISM OF NANOTOXICITY
Reprinted from Tyurnia et al. (2011) ACS Nano, published online Aug. 4, DOI: 10.1021/nn202201j. Copyright 2011, American Chemical Society. As the interest in single walled carbon nanotubes (SWCNTs) for technological and biomedical applications continues to grow, so does the concern regarding their toxicity. Accumulating evidence suggests that inhalational exposure to these particles leads to pulmonary inflammation, granuloma formation, and eventual fibrosis. Because oxidative stress has been proposed to play a role in SWCNT toxicity, Tyurina et al. [(2011) ACSNano, published online Aug 4, DOI:10.1021/nn202201j] used a lipidomics approach to investigate lipid peroxidation in the lungs of mice exposed to this material. Tyurina et al. treated mice by whole body inhalation for 4 days (5 h/day) with SWCNTs at a concentration of 5 mg/m3. They confirmed that the SWCNTs induced a robust pulmonary inflammatory response, characterized by sequential recruitment of neutrophils followed by macrophages and formation of persistent granulomas. Two-dimensional high-performance TLC of phospholipids from the lungs of control and SWCNT-treated mice at 1, 7, and 28 days following particle exposure demonstrated no significant change in the absolute quantities of lipids in the major phospholipid classes. However, lipidomic analysis using two-dimensional LC-MS and MALDI-TOF-MS revealed significant peroxidation of polyunsaturated fatty acid- (PUFA)-containing species of cardiolipin (CL), phosphatidylserine (PS), and phosphatidylinositol (PI). The most abundant oxidized fatty acid was linoleic acid. This result ruled out a random, nonspecific peroxidation event, which should have favored phosphatidylethanolamine and phosphatidylcholine since these lipids contain the bulk of the PUFAs that are the primary target of lipid peroxidation. Tyurina et al. note that cytochrome c can mediate the selective peroxidation of CL, PS, and PI under conditions of apoptosis. In fact, treatment of total lung lipids from control mice with cytochrome c and H2O2 led to a pattern of lipid peroxidation similar to that observed in the lungs of SWCNT-treated mice. Oxidized PS plays a role in apoptotic signaling and in the clearance of apoptotic cells. Oxidized CL facilitates the release of apoptotic mediators, including cytochrome c, from mitochondria. These results suggest a close relationship between pulmonary lipid peroxidation and apoptosis in response to SWCNTs and suggest that blockade of CL peroxidation in mitochondria may be an effective approach to protect against the toxic damage caused by SWCNT inhalation. Carol A. Rouzer
SPOTLIGHT
4-hydroxynonenal (HNE), and malondialdehyde (MDA). All of these compounds can react with proteins, leading to the formation of multiple adducts, some of which contain a reactive carbonyl (RC). Prior evidence has shown that RC-containing proteins can lead to activation of innate immunity, but relatively little is known about the adaptive immune response to these species, leading Moghaddam et al. [(2011) J. Immunol, 187, 1626] to investigate. Modification of hen egg lysozyme (HEL) with GA, MDA, or HNE led to the formation of RC adducts. Subsequent immunization of mice with the modified proteins induced an immune response as indicated by the generation of anti-HEL antibodies. The magnitude of the immune response was directly correlated with the RC content of the protein. Treatment of the modified proteins with NaBH4 to reduce the RCs to the corresponding alcohol markedly reduced their immunogenicity. Adaptive immune responses may be classified as Th1 or Th2. Th1 responses are characterized by the production of proinflammatory cytokines, notably interferon-γ and tumor necrosis factor-α, whereas Th2 responses are characterized by the production of the cytokines interleukin- (IL)-4, IL-5, and IL-13. Th2 responses play a role in the induction of humoral immunity and hypersensitivity reactions such as allergy. The dominant immunoglobulin isotype produced in response to RC-containing HEL was IgG1, suggesting a Th2-biased immune response. Moghaddam et al. confirmed this Th2-bias to RC-modified HEL in multiple mouse strains, even those more prone to Th1-biased responses. Dendritic cells play a key role in adaptive immunity through their ability to process antigens and present them to T cells. Moghaddam et al. showed that incubation of splenic dendritic cells with RC-adducted ovalbumin (OVA) increases their ability to stimulate a proliferative response in CD4+ T cells from transgenic mice bearing the OVA T cell receptor. The proliferative response induced by RC-adducted OVA in this assay was greater than that induced by unmodified OVA or RC-adducted ovalbumin treated with NaBH4. CD4+ T cells that proliferated in response to RC-adducted OVA produced greater quantities of the Th2-related cytokines IL-4 and IL-13 than T cells proliferating in response to unmodified or RC-adducted and reduced OVA. These results indicate that RC adducts enhance the dendritic cell-mediated presentation of a protein antigen, particularly to Th2-biased T cells. However, RC-adducted OVA was similar to unmodified OVA in its capacity to activate dendritic cells as indicated by the expression of surface markers or secretion of cytokines. Together, the data confirm a role for RCs in the promotion of adaptive immunity to damaged exogenous proteins. The Th2biased response induced by RC-modified proteins may serve both pathogenic and protective roles under conditions of oxidative stress. Further work will be required to more clearly define these roles and to determine if RCs promote an immune response to damaged endogenous proteins as well. Carol A. Rouzer
’ ADAPTIVE IMMUNITY TO OXIDATIVE STRESS Oxidative damage to carbohydrates and lipids leads to the production of reactive aldehydes, including glycolaldehyde (GA), 1600
dx.doi.org/10.1021/tx2003883 |Chem. Res. Toxicol. 2011, 24, 1599–1600
