SPOTLIGHT pubs.acs.org/crt
Nrf2-PROMOTED CARCINOGENESIS Ectopic overexpression of some oncogenes results in increased production of reactive oxygen species (ROS). This observation has led to the hypothesis that oxidative stress promotes carcinogenesis. However, as reported by DeNicola et al. [(2011) Nature, 475, 106], endogenous expression of the oncogenes K-RasG12D or MycERT2 leads to reduced H2O2, O2 , mitochondrial ROS, and 8-oxo-dGuo DNA adducts, accompanied by an increase in intracellular GSH/GSSG. This overall decrease in oxidant tone could be attributed to an increase in the expression and DNA binding activity of the transcription factor Nrf2 (Nfe2l2) in the oncogene-expressing cells. Nrf2, which is activated under conditions of oxidative stress, induces the expression of antioxidant genes, including Hmox1, Nquo1, Gclc, Gclm, and Ggt1. These genes were all up-regulated in cells expressing endogenous K-RasG12D, and knockdown of Nrf2 prevented the reduction in oxidant levels observed in response to endogenous K-RasG12D expression. A specific inhibitor of the kinase MEK restored Nrf2-dependent signaling and oxidant tone in K-RasG12D-expressing cells to levels observed in control cells, suggesting a role for MEK-dependent signaling in K-RasG12D-dependent Nrf2 stimulation. Support for this hypothesis came from the findings that expression of B-RafV619E, a constitutively active upstream regulator of MEK, mimicked the effect of K-RasG12D expression. Similarly, RNAi-mediated knockdown of the transcription factor Jun, a downstream effector in the MEK signaling pathway, blocked the effects of K-RasG12D expression on Nrf2 signaling. Both Jun and Myc bind upstream of the promotor of Nfe2l1, explaining how expression of K-RasG12D, MycERT2, and BRafV619E could all increase Nrf2 protein levels and antioxidant signaling. DeNicola et al. demostrated increased expression of the Nrf2 target gene Nqo1 and decreased immunoreactivity for 8-oxo-dGuo in mutant K-Ras-expressing murine and human pancreatic ductal carcinoma and murine lung adenomas. Nrf2 ablation reduced tumorigenicity in murine models of K-Ras-dependent pancreatic and lung carcinomas. Together the data support a role for increased Nrf2 signaling in carcinogenesis driven by some oncogenes. This finding contradicts the generally accepted paradigm that an antioxidant state is protective against cancer. Carol A. Rouzer ’ MECHANISM OF GLUCOSE TOXCITY
Growing evidence suggests that toxicity associated with high concentrations of glucose contributes to the vascular, renal, and ocular complications of diabetes. Glucose can react directly with the lysine residues of proteins to form a Shiff base, which following Amadori rearrangement, yields a stable adduct. Alternatively, oxidation of glucose generates reactive carbonyl species (RCS), including glyoxal (GO) and methylglyoxal (MGO), which may react with both the lysine and arginine residues of proteins. Now, Chetyrkin et al. [(2011) Biochemistry, 50, 6102] provide evidence that it is the latter reaction that is most detrimental to protein function. Prolonged incubation of lysozyme with glucose resulted in a concentration-dependent loss of enzymatic activity and modification of the protein as indicated by an altered LC retention time. MS analysis of the altered protein species indicated that r 2011 American Chemical Society
Amadori modifications were not associated with changes in enzyme activity but that GO or MGO adduction led to inactivation. Incubation of glucose alone under the same conditions generated both GO and MGO, and direct incubation of lysozyme with these RCS led to inactivation. Addition of metal chelators diethylene triamine pentaacetic acid (DTPA) or pyridoxamine (PM) blocked the generation of MGO and GO, and protected lysozyme during glucose exposure, suggesting that metal-catalyzed autoxidation of glucose was responsible for MGO and GO formation. MS analysis of glucose-exposed lysozyme confirmed the presence of multiple Amadori adducts at lysine residues as well as GO and MGO adducts at arginine residues, including arginine114, which is critical for enzyme activity. The RCS-dependent adducts, but not the Amadori adducts were prevented by the addition of PM. The RGD-R3 NC1 domain of collagen IV contains an arginine residue that is essential for the protein’s interaction with integrin. Chetyrkin et al. demonstrated that incubation of this protein with glucose inhibited binding to the integrin Rvβ3. Furthermore, the R1NC1 and R5NC1 domains of collagen IV isolated from the renal extracellular matrix of diabetic rats exhibited increased GO-derived modification of the critical arginine-169 when compared to that of protein from control rats. Chetyrkin et al. argue that because many proteins contain active site arginine residues, RCS modifications at this amino acid are likely to play a greater role in glucose-mediated toxicity associated with diabetes than the Amadori adducts obtained by direct reaction with glucose at lysine residues. Carol A. Rouzer
Published: September 19, 2011 1343
dx.doi.org/10.1021/tx200317c | Chem. Res. Toxicol. 2011, 24, 1343–1344
Chemical Research in Toxicology
’ HPNE-MEDIATED PROTEIN DAMAGE
Oxidation of polyunsaturated fatty acids (PUFAs) leads to the formation of reactive hydroperoxides and R,β-unsaturated aldehydes that can damage biomolecules, including proteins and nucleic acids. Among the best characterized of these reactive species are 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are derived from 4-hydroperoxy-2-nonenal (HPNE). Because HPNE is highly unstable, most investigators have assumed that it does not directly contribute to the cellular damage resulting from lipid peroxidation. However, Shimozu et al. [(2011) J. Biol. Chem., published online Jun 20, DOI 10.1074/jbc.M111.255737] now demonstrate the formation of HPNE-specific protein adducts in vitro and in vivo. Incubation of HPNE with bovine serum albumin (BSA) led to the disappearance of histidine and lysine residues from the protein. To identify the lysine reaction products, Shimozu et al. incubated HPNE with NR-benzoylglycyllysine. MS analysis of the reaction mixture revealed four products, I through IV, which were characterized by a combination of MS and NMR (see figure). To obtain monoclonal antibodies directed against HPNEmodified proteins, Shimozu et al. immunized mice with HPNEtreated keyhole limpet hemocyanin. Selection of antibodies that recognized HPNE-modified BSA but not the HNE- or ONEmodified protein yielded mAb PM9. Binding inhibition studies revealed that lysine adduct II but not adducts I, III, or IV could block the interaction of mAb PM9 with HPNE-modified BSA. This finding suggested that the antibody was directed against the structure of adduct II. When ω-6 PUFAs were reacted with Fe2+/ascorbate in the presence of BSA, mAb PM9-reactive protein adducts formed. This did not occur when ω-3 PUFAs were used. Similarly, mAb PM9 detected HPNE-lysine adducts in low density lipoproteins following oxidation with Cu2+. These results suggested that the formation of adduct II could occur under conditions of PUFA oxidation in the presence of proteins. The pathologic significance of the adducts was revealed by immunohistologic staining of atherosclerotic plaques from human patients. This approach revealed mAb PM9-immunoreactive material in vascular endothelial cells, macrophages, and migrating vascular smooth muscle cells. Staining did not occur in control vessel walls. Shimozu et al. conclude that HPNE does not simply serve as a precursor molecule for the generation of HNE and ONE. Rather, it is capable of directly damaging proteins, leading to the unique lysine adduct II. Further studies will demonstrate the contribution of HPNE-mediated protein damage to oxidant-dependent toxicity. Carol A. Rouzer
SPOTLIGHT
Biallelic mutation of any one of these genes leads to Fanconi anemia, a disease characterized by developmental defects, progressive bone marrow failure, and a predisposition to cancer. To better understand the specific kinds of DNA damage that are repaired by the Fanconi anemia pathway, Langevin et al. [(2011) Nature, 475, 53] explored the effect of disruption of the pathway on acetaldehyde-induced DNA damage. Disruption of the Fanconia anemia genes FANCL, FANCB, FANCC, FANCJ, and RAD51C in the DT40 chicken B cell line led to increased sensitivity to acetaldehyde toxicity. This did not occur when key proteins of other major DNA repair pathways were disrupted, suggesting that the Fanconia anemia pathway plays a unique role in the repair of acetaldehyde-mediated DNA damage. The enzyme aldehyde dehydrogenase 2 (Aldh2) is primarily responsible for the catabolism of acetaldehyde. Langevin et al. created mice deficient in this enzyme Aldh2 / and/or the Fanconia anemia pathway (Fancd2 / ) to explore the role of the pathway in mitigating acetaldehyde-induced DNA damage in vivo. Since all Fancd2 / mice were sterile, breeding experiments required heterozygotes for that gene. Langevin et al. first discovered that Aldh2 / Fanc2d( female mice crossed with Aldh2 / Fanc2d( or Aldh2( Fanc2d( male mice produced no offspring deficient in both genes, indicating that, in the case of Fancd2 / fetuses, either the mother or the fetus must have Aldh2 to catabolize acetaldehyde. When Aldh2 / Fancd2( males were crossed with Aldh2( Fancd2( females, exposure of the mother to ethanol at day E7 resulted in a decrease in double mutant offspring to 2.9% from 14.5% in saline-exposed controls. This decrease in double negative mutants was attributed to massive developmental defects. Ethanol is primarily metabolized to acetaldehyde. Thus, these results indicate that the absence of the Fanconia anemia pathway markedly increases sensitivity to ethanol and acetaldehyde toxicity during development. When 6 to 8 week old double mutant mice were exposed for 5 days to 15% ethanol followed by 5 days to 20% ethanol in their drinking water, they exhibited a marked decrease in all three blood constituents, attributable to an equally profound decrease in bone marrow cellularity and colony forming units. Bone marrow cells from ethanol-treated double mutants also showed increased levels of γH2AX, a marker of DNA damage, when compared to bone marrow from control double mutant or ethanol-treated wild-type mice. These results indicate that increased sensitivity to acetaldehyde persists into adulthood in Fanconia anemia mice. Double mutant mice showed only subtle developmental defects at birth, and they appeared healthy until the age of 3 to 6 months at which time many developed and ultimately succumbed to an illness that closely resembled acute lymphocytic leukemia. This observation suggests that failure to metabolize acetaldehyde hastens the leukemogenic phenotype of Fanconia’s anemia. Langevin et al. conclude that acetaldehyde-induced DNA damage may play an important role in the pathogenesis of Fanconia anemia, as well as contributing to fetal alcohol syndrome in healthy humans. Carol A. Rouzer
’ DEFENSE AGAINST ENDOGENOUS TOXICANTS The Fanconi anemia pathway mediates DNA repair through the complex interaction of proteins encoded by 14 different genes. 1344
dx.doi.org/10.1021/tx200317c |Chem. Res. Toxicol. 2011, 24, 1343–1344
