Dirk Lebrecht

Time-Dependent and Tissue-Specific Accumulation of mtDNA and Respiratory Chain Defects in Chronic Doxorubicin Cardiomyopathy

Background—Doxorubicin causes a chronic cardiomyopathy of unknown pathogenesis. We investigated whether acquired defects in mitochondrial DNA (mtDNA) and interconnected respiratory chain dysfunction may represent a molecular mechanism for its late onset. Methods and Results—Rats were treated weekly with intravenous doxorubicin (1 mg/kg) for 7 weeks, starting at 11 weeks of age (group B). Controls received saline. Group C received doxorubicin identically to group B, but the course was started at 41 weeks of age. All rats were killed at week 48. Doxorubicin was also injected once, either 6 days (group D) or 2 hours (group E) before euthanasia. Heart and skeletal muscle were examined. Only group B rats developed a significant clinical, macroscopic, histological, and ultrastructural cardiomyopathy. Group B hearts had the lowest cytochrome c oxidase (COX) activity (24% of controls; P =0.003), the highest citrate synthase activity (135% of controls; P =0.005), and the highest production of superoxide. In group B, the respiratory subunit COXI, which is encoded by mtDNA, was reduced (P <0.001), as was mtDNA (49% of controls, P <0.001). Group C hearts differed from group B in their lower cardiomyopathy score (P =0.006), higher COX activity (P =0.02), and higher mtDNA content (P =0.04). Group B and to a lesser extent group C hearts contained deleted mtDNA. There was no detectable mitochondrial toxicity in group D and E hearts or in skeletal muscle. Conclusions—In doxorubicin cardiomyopathy, mtDNA alterations, superoxide, and respiratory chain dysfunction accumulate long-term in the absence of the drug and are associated with a late onset.

Genetic CD21 deficiency is associated with hypogammaglobulinemia

BACKGROUND Complement receptor 2 (CR2/CD21) is part of the B-cell coreceptor and expressed by mature B cells and follicular dendritic cells. CD21 is a receptor for C3d-opsonized immune complexes and enhances antigen-specific B-cell responses. OBJECTIVE Genetic inactivation of the murine CR2 locus results in impaired humoral immune responses. Here we report the first case of a genetic CD21 deficiency in human subjects. METHODS CD21 protein expression was analyzed by means of flow cytometry and Western blotting. CD21 transcripts were quantified by using real-time PCR. The CD21 gene was sequenced. Wild-type and mutant CD21 cDNA expression was studied after transfection of 293T cells. Binding of EBV-gp350 or C3d-containing immune complexes and induction of calcium flux in CD21-deficient B cells were analyzed by means of flow cytometry. Antibody responses to protein and polysaccharide vaccines were measured. RESULTS A 28-year-old man presented with recurrent infections, reduced class-switched memory B cells, and hypogammaglobulinemia. CD21 receptor expression was undetectable. Binding of C3d-containing immune complexes and EBV-gp350 to B cells was severely reduced. Sequence analysis revealed a compound heterozygous deleterious mutation in the CD21 gene. Functional studies with anti-immunoglobulin- and C3d-containing immune complexes showed a complete loss of costimulatory activity of C3d in enhancing suboptimal B-cell receptor stimulation. Vaccination responses to protein antigens were normal, but the response to pneumococcal polysaccharide vaccination was moderately impaired. CONCLUSIONS Genetic CD21 deficiency adds to the molecular defects observed in human subjects with hypogammaglobulinemia.

Tissue-specific mtDNA lesions and radical-associated mitochondrial dysfunction in human hearts exposed to doxorubicin.

Doxorubicin causes a chronic cardiomyopathy. Although the exact pathogenesis is unknown, recent animal data suggest that somatically acquired alterations of mitochondrial DNA (mtDNA) and concomitant mitochondrial dysfunction play an important role in its onset. In this study, skeletal and myocardial muscles were examined from human autopsies. Compared to controls (n = 8), doxorubicin‐exposed hearts (n = 6) showed low absolute enzyme activity of mtDNA‐encoded nicotinamide adenine dinucleotide hydrogen dehydrogenase (NADH DH, 79% residual activity, p = 0.03) and cytochrome c oxidase (COX, 59% residual activity, p < 0.001), but not of succinate dehydrogenase (SDH), which is encoded exclusively by nuclear DNA. NADH DH/SDH and COX/SDH ratios were 37% (p < 0.001) and 27% (p < 0.001) of controls. Expression of the mtDNA‐encoded subunit II of COX was reduced (82%, p = 0.04), compared to its unchanged nucleus‐encoded subunit IV. MtDNA‐content was diminished (56%, p = 0.02), but the ‘common’ mtDNA‐deletion was increased (9.2‐fold, p = 0.004). Doxorubicin‐exposed hearts harboured numerous additional mtDNA rearrangements lacking direct repeats. They contained elevated levels of malondialdehyde (MDA) (p = 0.006, compared to controls), which correlated inversely with the COX/SDH ratio (r = −0.45, p = 0.02) and the mtDNA‐content (r = −0.75, p = 0.002), and correlated positively with the levels of the ‘common’ deletion (r = 0.80, p < 0.001). Doxorubicin‐exposed hearts also contained the highest levels of superoxide (p < 0.001, compared to controls), which correlated negatively with the mtDNA‐encoded respiratory chain activities, such as the COX/SDH ratio (r = −0.57, p = 0.02) and the NADH/SDH ratio (r = −0.52, p = 0.04), as well as with the mtDNA content (r = −0.69, p = 0.003), and correlated positively with the frequency of the ‘common’ deletion (r = 0.76, p < 0.001) and the MDA levels (r = 0.86, p < 0.001). Doxorubicin‐exposed hearts contained electron‐dense deposits within mitochondria. Hearts exposed to other anthracyclines (n = 6) or skeletal muscle (all groups) had no mitochondrial dysfunction. Doxorubicin, unlike other anthracyclines, augments lipid peroxidation, induces mtDNA mutations and decreases mtDNA content in human hearts. These lesions have an impact on mitochondrial function and could be of importance in the pathogenesis of clinical cardiomyopathy. Copyright

Impact of rituximab on immunoglobulin concentrations and B cell numbers after cyclophosphamide treatment in patients with ANCA-associated vasculitides.

Objective To assess the impact of immunosuppressive therapy with cyclophosphamide (CYC) and rituximab (RTX) on serum immunoglobulin (Ig) concentrations and B lymphocyte counts in patients with ANCA-associated vasculitides (AAVs). Methods Retrospective analysis of Ig concentrations and peripheral B cell counts in 55 AAV patients. Results CYC treatment resulted in a decrease in Ig levels (median; interquartile range IQR) from IgG 12.8 g/L (8.15-15.45) to 9.17 g/L (8.04-9.90) (p = 0.002), IgM 1.05 g/L (0.70-1.41) to 0.83 g/L (0.60-1.17) (p = 0.046) and IgA 2.58 g/L (1.71-3.48) to 1.58 g/L (1-31-2.39) (p = 0.056) at a median follow-up time of 4 months. IgG remained significantly below the initial value at 14.5 months and 30 months analyses. Subsequent RTX treatment in patients that had previously received CYC resulted in a further decline in Ig levels from pre RTX IgG 9.84 g/L (8.71-11.60) to 7.11 g/L (5.75-8.77; p = 0.007), from pre RTX IgM 0.84 g/L (0.63-1.18) to 0.35 g/L (0.23-0.48; p<0.001) and from pre RTX IgA 2.03 g/L (1.37-2.50) to IgA 1.62 g/L (IQR 0.84-2.43; p = 0.365) 14 months after RTX. Treatment with RTX induced a complete depletion of B cells in all patients. After a median observation time of 20 months median B lymphocyte counts remained severely suppressed (4 B-cells/µl, 1.25-9.5, p<0.001). Seven patients (21%) that had been treated with CYC followed by RTX were started on Ig replacement because of severe bronchopulmonary infections and serum IgG concentrations below 5 g/L. Conclusions In patients with AAVs, treatment with CYC leads to a decline in immunoglobulin concentrations. A subsequent RTX therapy aggravates the decline in serum immunoglobulin concentrations and results in a profoundly delayed B cell repopulation. Surveying patients with AAVs post CYC and RTX treatment for serum immunoglobulin concentrations and persisting hypogammaglobulinemia is warranted.

Role of mtDNA lesions in anthracycline cardiotoxicity

Doxorubicin (adriamycin) is an effective drug in the treatment of many malignancies. Its prolonged use is, however, limited by an irreversible, dose-dependent and progressive cardiomyopathy, which may become evident even years after completion of therapy. Data from rats and humans show that oxidative phosphorylation is impaired rapidly after acute doxorubicin-exposure. Such respiratory chain dysfunction is known to enhance the production of reactive oxygen species and may lead to quantitative and qualitative injury of mitochondrial DNA (mtDNA) and its encoded respiratory chain subunits. MtDNA depletion, mtDNA mutations and respiratory defects then accumulate with time also in the absence of continued anthracycline exposure. Chronic cardiotoxicity then manifests, when the bioenergetic capacity of the organelles is severely impaired. The mitochondrial damage in late-onset doxorubicin cardiomyopathy is heart specific and not found in skeletal muscle. DOXO-EMCH, a 6-maleimidocaproyl hydrazone derivative of doxorubicin has evolved from the search for less cardiotoxic anthracyclines. At equieffective antitumor doses, DOXO-EMCH has a substantially lower heart toxicity than free doxorubicin.

Mitochondrial Tubulopathy in Tenofovir Disoproxil Fumarate-Treated Rats

Objectives:Tenofovir disoproxil fumarate (tenofovir DF) use has been associated with renal dysfunction and Fanconi syndrome. Tenofovir is taken up into renal tubules by anion transporters where high intracellular drug concentration may induce a functionally relevant depletion of mitochondrial DNA (mtDNA). We investigated if tenofovir may induce renal mtDNA depletion and respiratory chain dysfunction. Methods:Rats (n = 8) were gavaged daily with 100 mg·kg−1·d−1 of tenofovir DF or didanosine. Kidneys and livers were examined after 8 weeks of treatment. Results:The tenofovir group had significantly lower body and kidney weights than rats exposed to water or didanosine. Proximal but not distal tubules were of increased diameter and contained small lipid droplets. Tubular mitochondria were enlarged, and their crystal architecture was disrupted. Tenofovir-exposed kidneys contained low mtDNA copy numbers and impaired expression of mtDNA-encoded cytochrome c oxidase (COX) I but not nucleus-encoded COX IV subunits. Histochemistry demonstrated low tubular COX and nicotinamide adenine dinucleotide dehydrogenase (NADH-DH) activities, whereas succinate dehydrogenase activity was preserved. COX activity was preserved in the glomeruli of tenofovir-exposed rats. Didanosine did not elicit renal effects but, unlike tenofovir, depleted mtDNA in liver (by 52%). Conclusions:Tenofovir DF induces an organ-specific nephrotoxicity with mtDNA depletion and dysfunction of mtDNA-encoded respiratory chain subunits. The data do not support nephrotoxicity of didanosine.

Effect of Reducing the Dose of Stavudine on Body Composition, Bone Density, and Markers of Mitochondrial Toxicity in HIV-Infected Subjects: A Randomized, Controlled Study

BACKGROUND Stavudine is widely used in developing countries. Lipoatrophy and mitochondrial toxicity have been linked to stavudine use, but it is unclear whether switching to a lower dose can reduce these toxicities while maintaining human immunodeficiency virus (HIV) suppression. METHODS HIV-infected subjects receiving standard-dose stavudine with undetectable HIV type 1 RNA for > or =6 months were randomized (ratio, 3:2) to receive one-half of the stavudine dose (switch arm) or to maintain the dose (continuation arm) while continuing to receive all other prescribed antiretrovirals. The following measurements were obtained at baseline and week 48: fasting lactate, pyruvate, and lipid levels; results of whole-body dual-energy x-ray absorptiometry; and mitochondrial DNA (mtDNA) measurements in fat and peripheral blood mononuclear cells. Change from baseline to week 48 was compared within and between groups. RESULTS Twenty-four patients (79% of whom were men and 79% of whom were African American; median age, 45 years) were enrolled in the study, 15 were enrolled in the switch arm, and 9 were enrolled in the continuation arm. The median duration of stavudine treatment was 55 months (range, 21-126 months). The median CD4 cell count was 558 cells/mm(3) (range, 207-1698 cells/mm(3)). At baseline, the study arms had similar demographic characteristics and laboratory indices, except for body mass index, total lean body mass, and triglyceride levels (all of which were higher in the switch arm). Three patients (2 in the switch arm) discontinued the study because of study-unrelated reasons. CD4 cell counts remained unchanged. At 48 weeks, 6 patients (4 [27%] in the switch arm and 2 [22%] in the continuation arm) had detectable HIV RNA levels (median, 972 copies/mL; range, 60-49,400 copies/mL). All patients with detectable HIV RNA levels reported significant lapses in treatment adherence; none exhibited mutations in HIV genotype. After the treatment switch, significant changes from study entry to week 48 were noted only for lactate level (median change, -0.27 mmol/L; range, -1.2 to 0.25 mmol/L; P = .02) and fat mtDNA (median change, 40 copies/cell; range, -49 to 261 copies/cell; P = .02). In the continuation arm, a significant loss of bone mineral density was seen at week 48 (median change, -1.7%; range, -6.3% to 0.8%; P = .02). The only significant between-group difference was the change in bone mineral density from baseline (P = .003). CONCLUSIONS Reducing stavudine dose by one-half increased fat mtDNA and decreased lactate levels, suggesting improvement in mitochondrial indices while preserving HIV suppression in subjects who maintained adherence. A significant loss of bone mineral density was seen in patients receiving standard-dose stavudine but not in those receiving low-dose stavudine. These results suggest that switching to low-dose stavudine may improve mitochondrial indices while maintaining virological suppression.

The 6-maleimidocaproyl hydrazone derivative of doxorubicin (DOXO-EMCH) is superior to free doxorubicin with respect to cardiotoxicity and mitochondrial damage.

Doxorubicin causes a chronic cardiomyopathy in which genetic and functional lesions of mitochondria accumulate in the long‐term and explain in part the delayed onset of heart dysfunction. DOXO‐EMCH a 6‐maleimidocaproyl hydrazone derivative of doxorubicin, is an albumin binding prodrug which has entered clinical trials because of its superior antitumor and toxicological profile. In the present work, we examined the chronic cardiotoxicity of DOXO‐EMCH in direct comparison with doxorubicin. Rats (11 weeks of age) were treated with intravenous doxorubicin (0.8 mg/kg weekly for 7 weeks), an equimolar dose of DOXO‐EMCH (1.1 mg/kg), or with 3.3 mg/kg of DOXO‐EMCH. Controls received saline. Animals were euthanized at 48th week. Rats exposed to doxorubicin had a severe clinical, and histopathological cardiomyopathy with depressed myocardial activity of cytochrome c‐oxidase (COX, 26% of controls), reduced expression of the mtDNA‐encoded COX II subunit, decreased mtDNA copy numbers (46% of controls), and high levels of malondialdehyde and superoxide (787% of controls). All parameters were highly correlated with myocardial damage. Both DOXO‐EMCH groups did not differ from controls with regard to clinical symptomatology, mortality and mitochondrial enzymes, although the myocardia of the high‐dose group had slightly increased histopathological abnormalities, depressed mtDNA copies (74% of controls) and elevated superoxide levels (347% of controls). Doxorubicin‐exposed hearts and to a lesser extent the myocardia of both DOXO‐EMCH groups contained mtDNA‐deletions. In summary both DOXO‐EMCH doses were superior over doxorubicin with respect to clinical and histopathological evidence of cardiomyopathy, myocardial COX‐activity, COX II expression, mtDNA‐content, mtDNA mutation loads and superoxide production in rats.

Pyrimidine Nucleoside Depletion Sensitizes to the Mitochondrial Hepatotoxicity of the Reverse Transcriptase Inhibitor Stavudine

Stavudine is a hepatotoxic antiretroviral nucleoside analogue that also inhibits the replication of mitochondrial DNA (mtDNA). To elucidate the mechanism and consequences of mtDNA depletion, we treated HepG2 cells with stavudine and either redoxal, an inhibitor of de novo pyrimidine synthesis, or uridine, from which pyrimidine pools are salvaged. Compared with treatment with stavudine alone, co-treatment with redoxal accelerated mtDNA depletion, impaired cell division, and activated caspase 3. These adverse effects were completely abrogated by uridine. Intracellular ATP levels were unaffected. Transcriptosome profiling demonstrated that redoxal and stavudine acted synergistically to induce CDKN2A and p21, indicating cell cycle arrest in G1, as well as genes involved in intrinsic and extrinsic apoptosis. Moreover, redoxal and stavudine showed synergistic interaction in the up-regulation of transcripts encoded by mtDNA and the induction of nuclear transcripts participating in energy metabolism, mitochondrial biogenesis, oxidative stress, and DNA repair. Genes involved in nucleotide metabolism were also synergistically up-regulated by both agents; this effect was completely antagonized by uridine. Thus, pyrimidine depletion sensitizes cells to stavudine-mediated mtDNA depletion and enhances secondary cell toxicity. Our results indicate that drugs that diminish pyrimidine pools should be avoided in stavudine-treated human immunodeficiency virus patients. Uridine supplementation reverses this toxicity and, because of its good tolerability, has potential clinical value for the treatment of side effects associated with pyrimidine depletion.

Increased mtDNA levels without change in mitochondrial enzymes in peripheral blood mononuclear cells of infants born to HIV-infected mothers on antiretroviral therapy.

Abstract Background: The effects of gestational nucleoside reverse transcriptase inhibitors (NRTIs) on mitochondrial DNA (mtDNA) are controversial. The effects of mtDNA depletion on mitochondrial function have not been assessed. Method: In peripheral blood mononuclear cells (PBMCs) from infants born to HIV-infected women and infants born to HIV-1-uninfected women, mtDNA copy numbers were determined by quantitative PCR; nuclear (COXIV)- and mitochondrial (COXII)-encoded polypeptides of the oxidative phosphorylation enzyme cytochrome c-oxidase (COX or complex IV) were quantified by Western blot. Results: Overall, 86 infants born to HIV-infected women and 50 controls were studied. HIV-infected mothers had a median CD4 count of 506 cells/μL; 59% had HIV RNA ≤ 50 copies/mL. No infant had clinical evidence of mitochondrial disease. The birth weight was lower (p = .016) and the body length higher (p = .002) in the HIV-exposed newborns. Eighty-one HIV-infected women had received gestational NRTIs (median duration 162 days). Median mtDNA copies/PBMC in the HIV-exposed infants were 505 (range, 120-1365) vs. 213 (27-426) in controls (p < .001). COX II/IV ratios were similar in both groups. Although mtDNA levels correlated inversely with maternal lactate, mitochondrial indices did not correlate with maternal CD4+ count, HIV RNA, smoking, or alcohol consumption. Conclusion: We found elevated mtDNA copy numbers in PBMC of infants born to HIV-infected women, the majority of whom received NRTI-based therapy, when compared to those born to healthy HIV-negative controls, but there was no difference in mtDNA-encoded respiratory chain protein. The clinical consequence of these findings is unknown and requires further investigations.