Hematological indices of injury to lightly oiled birds from the Deepwater Horizon oil spill.

Avian mortality events are common following large-scale oil spills. However, the sublethal effects of oil on birds exposed to light external oiling are not clearly understood. We found that American oystercatchers (area of potential impact n = 42, reference n = 21), black skimmers (area of potential impact n = 121, reference n = 88), brown pelicans (area of potential impact n = 91, reference n = 48), and great egrets (area of potential impact n = 57, reference n = 47) captured between 20 June 2010 and 23 February 2011 following the Deepwater Horizon oil spill experienced oxidative injury to erythrocytes, had decreased volume of circulating erythrocytes, and showed evidence of a regenerative hematological response in the form of increased reticulocytes compared with reference populations. Erythrocytic inclusions consistent with Heinz bodies were present almost exclusively in birds from sites impacted with oil, a finding pathognomonic for oxidative injury to erythrocytes. Average packed cell volumes were 4 to 19% lower and average reticulocyte counts were 27 to 40% higher in birds with visible external oil than birds from reference sites. These findings provide evidence that small amounts of external oil exposure are associated with hemolytic anemia. Furthermore, we found that some birds captured from the area impacted by the spill but with no visible oiling also had erythrocytic inclusion bodies, increased reticulocytes, and reduced packed cell volumes when compared with birds from reference sites. Thus, birds suffered hematologic injury despite no visible oil at the time of capture. Together, these findings suggest that adverse effects of oil spills on birds may be more widespread than estimates based on avian mortality or severe visible oiling. Environ Toxicol Chem 2018;37:451-461. © 2017 SETAC.

A novel flow cytometry-based method of analyzing Heinz bodies.

INTRODUCTION: Heinz bodies are important to diagnosing and managing patients. However, microscopic examination of Heinz bodies has several disadvantages, demonstrating the need for a better method. We explored the potential use of flow cytometry to examine Heinz bodies. METHODS: Whole-blood samples were collected from patients deficient in G6PD and healthy volunteers. Acetylphenylhydrazine was used to induce formation of Heinz bodies in red blood cells (RBCs). Then, RBCs positive for Heinz bodies were examined using a FACSCanto II cytometer. RESULTS: RBCs treated with acetylphenylhydrazine formed Heinz bodies and emitted a broad spectrum of fluorescence that could be detected by flow cytometry. The maximum emission of fluorescence was observed at 45 min after the incubation with acetylphenylhydrazine. In addition, the fluorescence emitted was stable for at least 72 h. The flow cytometer could detect the RBCs positive for Heinz bodies even if they made up as little as 0.1% of the total RBC population. Furthermore, the percentage and number, respectively, of RBCs positive for Heinz bodies in G6PD-deficient patients and normal donors exhibited a mean ± standard deviation (SD) of 68.9 ± 27.5 vs. 50.9 ± 28.6 and 96 014 ±35 732 cells/µL vs. 74 688 ± 36 514 cells/µL. CONCLUSION: Heinz bodies induced by acetylphenylhydrazine emit fluorescence, and this fluorescence could be examined using flow cytometry. Our study suggests the potential use of the developed method to investigate the formation of Heinz bodies in clinical samples.

Clinical oxidative stress during leprosy multidrug therapy: impact of dapsone oxidation.

This study aims to assess the oxidative stress in leprosy patients under multidrug therapy (MDT; dapsone, clofazimine and rifampicin), evaluating the nitric oxide (NO) concentration, catalase (CAT) and superoxide dismutase (SOD) activities, glutathione (GSH) levels, total antioxidant capacity, lipid peroxidation, and methemoglobin formation. For this, we analyzed 23 leprosy patients and 20 healthy individuals from the Amazon region, Brazil, aged between 20 and 45 years. Blood sampling enabled the evaluation of leprosy patients prior to starting multidrug therapy (called MDT 0) and until the third month of multidrug therapy (MDT 3). With regard to dapsone (DDS) plasma levels, we showed that there was no statistical difference in drug plasma levels between multibacillary (0.518±0.029 µg/mL) and paucibacillary (0.662±0.123 µg/mL) patients. The methemoglobin levels and numbers of Heinz bodies were significantly enhanced after the third MDT-supervised dose, but this treatment did not significantly change the lipid peroxidation and NO levels in these leprosy patients. In addition, CAT activity was significantly reduced in MDT-treated leprosy patients, while GSH content was increased in these patients. However, SOD and Trolox equivalent antioxidant capacity levels were similar in patients with and without treatment. These data suggest that MDT can reduce the activity of some antioxidant enzyme and influence ROS accumulation, which may induce hematological changes, such as methemoglobinemia in patients with leprosy. We also explored some redox mechanisms associated with DDS and its main oxidative metabolite DDS-NHOH and we explored the possible binding of DDS to the active site of CYP2C19 with the aid of molecular modeling software.

A practical quantification method for Heinz bodies in birds applicable to rapid response field scenarios.

Oil-induced oxidative injury to red blood cells results in Heinz body hemolytic anemia. Here, we evaluated three Heinz body staining techniques in brown pelican (Pelecanus occidentalis) blood. Using a range of in vitro acetylphenylhydrazine incubations, we validated a field-adapted technique against laboratory wet-mounts and verified the stability of this technique for one month following preparation. Employing this technique during petrochemical spill responses allows for delays between sample collection and analysis.

Peroxiredoxin I deficiency attenuates phagocytic capacity of macrophage in clearance of the red blood cells damaged by oxidative stress.

The role of peroxiredoxin (Prx) I as an erythrocyte antioxidant defense in red blood cells (RBCs) is controversial. Here we investigated the function of Prx I by using Prx I(-/-) and Prx I/II(-/-) mice. Prx I(-/-) mice exhibited a normal blood profile. However, Prx I/II(-/-) mice showed more significantly increased Heinz body formation as compared with Prx II(-/-) mice. The clearance rate of Heinz body-containing RBCs in Prx I(-/-) mice decreased significantly through the treatment of aniline hydrochloride (AH) compared with wild-type mice. Prx I deficiency decreased the phagocytic capacity of macrophage in clearing Heinz body-containing RBCs. Our data demonstrate that Prx I deficiency did not cause hemolytic anemia, but showed that further increased hemolytic anemia symptoms in Prx II(-/-) mice by attenuating phagocytic capacity of macrophage in oxidative stress damaged RBCs, suggesting a novel role of Prx I in phagocytosis of macrophage.

Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies.

The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species.

Molecular biosensing mechanisms in the spleen for the removal of aged and damaged red cells from the blood circulation.

Heinz bodies are intraerythrocytic inclusions of hemichrome formed as a result of hemoglobin (Hb) oxidation. They typically develop in aged red cells. Based on the hypothesis that hemichrome formation is an innate characteristic of physiologically normal Hb molecules, we present an overview of our previous findings regarding the molecular instability of Hb and the formation of hemichrome, as well as recent findings on Heinz body formation within normal human erythrocytes. Human adult Hb (HbO(2) A) prepared from healthy donors showed a tendency to produce hemichrome, even at close to physiological temperature and pH. Recent studies found that the number of Heinz bodies formed in red cells increased with increasing temperature when freshly drawn venous blood from healthy donors was subjected to mild heating above 37 °C. These findings suggest that Hb molecules control the removal of non-functional erythrocytes from the circulation via hemichrome formation and subsequent Heinz body clustering. In this review, we discuss the molecular biosensing mechanisms in the spleen, where hemichrome formation and subsequent Heinz body clustering within erythrocytes play a key role in the removal of aged and damaged red cells from the blood circulation.

Effect of vitamin C, deferoxamine, quercetin and rutin against tert-butyl hydroperoxide oxidative damage in human erythrocytes.

The mature human erythrocyte, when submitted to oxidative stress, can demonstrate depletion of reduced glutathione, oxidation of the hemoglobin molecule and aggregation of complexes of iron close to the membrane. These can produce abnormalities in the erythrocyte membrane and hemolysis. The aim of this work was to study the antioxidative action of vitamin C (vit. C), deferroxamine (DFO) and the flavonoids quercetin and rutin in normal human erythrocytes, submitted to in vitro oxidative stress induced by tert-butylhydroperoxide ((t)BHP). Venous blood was collected in citrate-phosphate-dextrose (CPD) solution, as anticoagulant, from healthy adult individuals after informed consent. The erythrocytes were resuspended in PBS to obtain 35% globular volume, and then submitted to the oxidative action of (t)BHP for up to 30 min, with or without previous incubation for 60 min with vit. C, DFO, quercetin and rutin. Decrease in the GSH concentration, G6-PD and GR activities, and increase in the methemoglobin and Heinz bodies (HB) formation, occurred with the increase in (t)BHP concentration. (t)BHP did not effect on the membrane proteins detected by SDS-PAGE. Quercetin, partially prevented the GSH decrease and the formation of HB, but did not prevent MetHb formation from oxidative damage by (t)BHP. Rutin, after (t)BHP induction, prevented the GSH decrease and the formation of HB. Vit. C, had no influence on the depletion of GSH, inhibited partially the metHb formation, and it protected GR, but not G6-PD from oxidative damage by (t)BHP. DFO partially inhibited the metHb formation and GSH decrease, but it did not protect GR and G6-PD from oxidative damage by (t)BHP. The results obtained suggest that vit. C, DFO and the flavonoids quercetin and rutin contribute to the decrease in the oxidative stress caused by (t)BHP.

The effects of superoxide dismutase knockout on the oxidative stress parameters and survival of mouse erythrocytes.

The erythrocytes of 12-month old Sod1 (-/-) mice showed an increased level of reactive oxygen species (ROS), as estimated by the degree of dihydroethidine and dihydrorhodamine oxidation, and the increased level of Heinz bodies. No indices of severe oxidative stress were found in the red blood cells and blood plasma of Sod1 (-/-) mice as judged from the lack of significant changes in the levels of erythrocyte and plasma glutathione, plasma protein thiol and carbonyl groups and thiobarbituric-acid reactive substances in the blood plasma. However, a decreased erythrocyte lifespan, increased reticulocyte count and splenomegaly were noted, indicating the importance of superoxide dismutase for maintaining erythrocyte viability. The levels of erythrocyte ROS and Heinz bodies and the reticulocyte count were indistinguishable in Sod1 (+/+) and Sod1 (+/-) mice, suggesting that a superoxide dismutase activity decrease to half of its normal value may be sufficient to secure the protective effects of the enzyme.

Oxidative process in erythrocytes of individuals with hemoglobin S.

The understanding of the oxidative stress mechanisms helps to explain many of the processes of cellular lesion and death, especially those related to the hemolytic diseases. Sickle cell anemia, thalassemias and G6-PD deficiency are among the more frequent genetic anomalies accompanied by oxidative stress. In the sickle cells, one of the factors that predisposes to the hemolytic process is the oxidative degradation of the hemoglobin S due to its deoxigenation leading to hemichrome formation and precipitation as Heinz bodies. The oxidative stress contributes to the sickle process and shortening of the erythrocyte survival. Here we analyzed the oxidative process in erythrocytes of patients with two different genotypes for HbS (AS and SS). Units of blood from donors of the Center of Hematology and Hemotherapy of Paraná (HEMEPAR), from normal individuals (AA) and from heterozygote individuals (AS), and venous blood collected from patients with sickle cell anemia (SS) were analyzed. In order to evaluate the protective action of the vitamins C and E in oxidative stress, erythrocytes were treated with antioxidant substances, vitamin C and vitamin E, and then treated with the oxidant tert-butilhydroperoxide (TBHP). The oxidative action induced by TBHP was observed in erythrocytes AA

Biomarkers of polycyclic aromatic hydrocarbon (PAH)-associated hemolytic anemia in oiled wildlife.

Polycyclic aromatic hydrocarbons (PAHs) in crude oil cause a range of adverse effects in oiled seabirds, one of the most common being hemolytic anemia via oxidative attack of erythrocytes by PAH metabolites resulting in hemoglobin leakage and formation of Heinz bodies. In such cases, haptoglobin and ferritin are up-regulated to sequester free Hb and iron in the circulation. We investigated these plasma proteins as biomarkers of PAH-induced Heinz body hemolytic anemia in oiled seabirds. Concentration ranges of PAHs, HAP and FT in plasma samples were 10-184 ng/ml, 0-2.6 mg/ml and 0-7.6 ng/ml, respectively. Dose-response relationships between plasma PAH exposure and haptoglobin and ferritin (FT) were investigated, and evidence of erythrocyte Heinz body formation studied in 50 oiled common guillemots stranded on the Norfolk Wash coast (East England). Haptoglobin was negatively correlated, and FT was positively correlated with PAH exposure. Heinz bodies were also observed confirming the toxic mechanism causing hemolytic anemia and counts were positively correlated with exposure. Our results support the application of these complementary biomarkers to assess hemolytic effects of oiling in wildlife biomonitoring, which also discriminate the influence of hemolytic versus inflammatory effects in oiled guillemots.

Heinz body haemolytic anaemia in a dog secondary to ingestion of a zinc toy: a case report.

A 6-year-old Labrador retriever was referred for investigation of severe lethargy and suspected immune-mediated haemolytic anaemia. Clinical examination revealed pale mucous membranes and jaundice. Haematology demonstrated large numbers of Heinz bodies and a marked anaemia, which was strongly regenerative. Serum zinc concentrations were markedly elevated. Analysis of a metal toy vomited by the dog 3 days prior to presentation revealed it to be composed of almost pure zinc. A diagnosis of haemolytic anaemia secondary to acute zinc toxicity was made and supportive therapy instigated. There was a subsequent decrease in numbers of Heinz bodies and a rise in the haematocrit, and the dog made an uneventful recovery. Acute zinc toxicity resulting in haemolytic anaemia is rarely observed, and this case was also unusual in that the main clinicopathological finding was the presence of numerous Heinz bodies without other evidence of oxidative damage to red blood cells.

Heinz body hemolytic anemia with eccentrocytosis from ingestion of Chinese chive (Allium tuberosum) and garlic (Allium sativum) in a dog.

A 4-year-old, intact male miniature schnauzer was presented with anorexia. The dog had ingested some Chinese steamed dumplings 2 days before, which contained Chinese chive (Allium tuberosum) and garlic (Allium sativum). Hematological examinations revealed severe Heinz body hemolytic anemia with eccentrocytosis and an increased concentration of methemoglobin, which was thought to result from oxidative damage to erythrocytes by constituents in these Allium plants. In this case, eccentrocytosis was a hallmark finding and could be detected easily, suggesting that this hematological abnormality is useful in diagnosing Allium plant-induced hemolysis.

Loss of alpha-hemoglobin-stabilizing protein impairs erythropoiesis and exacerbates beta-thalassemia.

Hemoglobin (Hb) A production during red blood cell development is coordinated to minimize the deleterious effects of free alpha- and beta-Hb subunits, which are unstable and cytotoxic. The alpha-Hb-stabilizing protein (AHSP) is an erythroid protein that specifically binds alpha-Hb and prevents its precipitation in vitro, which suggests that it may function to limit free alpha-Hb toxicities in vivo. We investigated this possibility through gene ablation and biochemical studies. AHSP(-/-) erythrocytes contained hemoglobin precipitates and were short-lived. In hematopoietic tissues, erythroid precursors were elevated in number but exhibited increased apoptosis. Consistent with unstable alpha-Hb, AHSP(-/-) erythrocytes contained increased ROS and evidence of oxidative damage. Moreover, purified recombinant AHSP inhibited ROS production by alpha-Hb in solution. Finally, loss of AHSP worsened the phenotype of beta-thalassemia, a common inherited anemia characterized by excess free alpha-Hb. Together, the data support a model in which AHSP binds alpha-Hb transiently to stabilize its conformation and render it biochemically inert prior to Hb A assembly. This function is essential for normal erythropoiesis and, to a greater extent, in beta-thalassemia. Our findings raise the possibility that altered AHSP expression levels could modulate the severity of beta-thalassemia in humans.

Two-generation reproductive study in mink fed diisopropyl methylphosphonate (DIMP).

Two generations of "Ranch Wild" mink (Mustela vison) were fed the organophosphate diisopropyl methylphosphonate (DIMP) at 0, 150, 450, or 1250ppm, to determine potential toxicity to the dams. Chemical, hematologic, necropsy, and microscopic examinations were performed on all parental animals and representative kits. The F0 and F1 dams had 3.4 and 4.6% mortality, respectively, distributed among all groups and not attributed to DIMP exposure. Adverse effects were mild and limited to the highest dose group. Plasma cholinesterase was reduced 40% (F0) and 31% (F1), as was whole blood cholinesterase (16 and 8.5%). Heinz bodies were present in 2.8% (F0) and 1.3% (F1) of erythrocytes. The erythrocyte count was reduced 6.3% in the F0. Reproductive efficiency was not affected. The mink were not uniquely susceptible to DIMP, relative to the literature on other species. The no observed adverse effect level (NOAEL), based on the 450ppm group of F1 females, was 56.5mg DIMP/kgBW per day; the lowest observed adverse effect level (LOAEL) was 329.5mg DIMP/kgBW per day.

The role of calpain and calpastatin in the catabolism of erythrocyte-membrane proteins during anaemia in hamsters (Mesocricetus auretus) infected with Leishmania donovani.

The anaemia associated with visceral leishmaniasis is accompanied by altered Ca(2+) homeostasis and degradation of the cytoskeletal and integral proteins of the erythrocytic membrane. In the present study, such changes were followed in hamsters that were anaemic as the result of their experimental infection with Leishmania donovani. At each stage of the infection, the blood concentration of haemoglobin was found to be negatively correlated with the concentration of Ca(2+) (R(2) = 0.91), the percentage of erythrocytes with Heinz bodies (R(2) = 0.98) and thiol depletion (R(2) = 0.96) in the erythrocytes. Calpain (Ca(2+)-activated protease; EC 3.4.22.17) and its natural inhibitor calpastatin are known to regulate the catabolism of membrane structural proteins. Densitometric scanning of SDS-PAGE gels showed that erythrocytic membranes from infected hamsters contained less calpain and calpastatin than those from control animals. The level of calpain autolysis was found to increase as the infection progressed. The addition of purified calpain (from control hamsters) to erythrocyte ghosts caused greater degradation of the membranes of erythrocytes from infected animals than of the corresponding membranes from control animals. Calpastatin from the control hamsters was more effective, at inhibiting calpain-induced membrane proteolysis, than calpastatin from the infected animals. The results indicate that the Ca(2+)-activated protease and its inhibitor are involved in the degradation of erythrocytic membranes observed during visceral leishmaniasis.

Heinz body formation in cats fed baby food containing onion powder.

OBJECTIVE: To determine whether cats fed baby food with onion powder develop Heinz bodies and anemia and to establish a dose-response relation between dietary onion powder content and Heinz body formation. DESIGN: Prospective study. ANIMALS: 42 healthy, adult, specific-pathogen-free cats. PROCEDURE: Commercial baby food with and without onion powder was fed to 2 groups of 6 cats for 5 weeks. Heinz body percentage, PCV, reticulocyte percentage, turbidity index, and methemoglobin and reduced glutathione concentrations were determined twice weekly and then weekly for 4 weeks following removal of the diet. For the dose-response study, 5 groups of 6 cats were fed a canned diet for 2 months that contained 0, 0.3, 0.75, 1.5, or 2.5% onion powder. Heinz body percentage, PCV, and reticulocyte percentage were determined twice weekly. RESULTS: Compared with cats fed baby food without onion powder, cats ingesting baby food with onion powder had significantly higher Heinz body percentages that peaked at 33 to 53%. Methemoglobin concentration also significantly increased but did not exceed 1.2%. Glutathione concentration, PCV, and food intake did not differ between the 2 groups. Rate and degree of Heinz body formation differed significantly between various onion powder concentrations fed. Compared with 0% onion powder, the diet with 2.5% onion powder caused a significant decrease in PCV and an increased punctate reticulocyte percentage. CLINICAL IMPLICATIONS: Baby food or other foods containing similar amounts of onion powder should be avoided for use in cats because of Heinz body formation and the potential for development of anemia, particularly with high food intake. Cats with diseases associated with oxidative stress may develop additive hemoglobin damage when fed baby food containing onion powder.

Oxidative damage by phenylhydrazine diminishes erythrocyte anion transport.

Human erythrocytes were exposed to oxidative stress by treatment with the slowly hemolytic drug phenylhydrazine. Phenylhydrazine has been previously shown to trigger the production of toxic oxygen metabolites including O-2 and H2O2 and the formation of Heinz bodies. The concentration-dependent formation of Heinz bodies was confirmed using optical microscopy. Heinz body formation was accompanied by surface protuberances as shown by scanning electron microscopy. The formation of Heinz bodies was accompanied by inhibition of anion translocation. Anion translocation was measured using the anionic fluorescent substrate analog N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine). The efflux of NBD-taurine was measured by continuous monitoring of transport by fluorescence (CMTF). The mean value of the kinetic rate constant for transport, k, was found to be -0.090 +/- 0.017 min-1. Phenylhydrazine was found to decrease k to less than one-half of control values in a dose-dependent fashion. The disruption of anion translocation may be related to the oxidative effects of phenylhydrazine and to the generation of Heinz bodies, which bind to the N-terminal domain of band 3.