Heinz Body Formation Research Articles

Effect of Garlic Extract on the Erythrocyte as a Simple Model Cell.

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl- and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation.

Comparison of alfaxalone and propofol on haematological and serum biochemical variables in cats undergoing radiotherapy with sevoflurane maintenance

ObjectiveTo evaluate effects of repeated alfaxalone or propofol administration on haematological and serum biochemical variables in cats undergoing radiotherapy. Study designProspective, block-randomized, clinical trial. AnimalsA group of 39 client-owned cats. MethodsAfter butorphanol (0.2 mg kg–1) and midazolam (0.1 mg kg–1) sedation, cats were randomly assigned to receive either alfaxalone or propofol for induction of anaesthesia and sevoflurane maintenance. Cats were anaesthetized daily with the same induction agent for 10–12 days. Complete blood counts, reticulocytes, Heinz body score and serum biochemistry were performed before the first treatment (T1), at T6, T10 and 3 weeks after the final treatment (T21). Cumulative induction agent dose for each cat at each time point was evaluated for an effect on Heinz body score. Data are shown as mean ± standard deviation; p < 0.05. ResultsAt baseline there were no significant differences in signalment or blood variables between groups. A significant decrease in haematocrit of 2.3% ± 0.77 (p = 0.02) between T1-T6 and T1-T10 [mean 4.1% (± 0.78, p < 0.0001)] was detected, with a significant increase in haematocrit of 2.1% ± 0.80 (p = 0.046) between T6-T21 and 4.0% ± 0.8 (p < 0.001) between T10-T21. Heinz body score significantly increased by 1.86 ± 0.616 (p = 0.013) between T1-T10. In the propofol group, reticulocytes increased significantly between T1-T6 [mean 23,090 μL–1 ± 7670 (p = 0.02)] and T1-T10 [mean 27,440 μL–1 ± 7990 (p = 0.007)]. Mean cumulative dose at T10 was 19.65 mg kg–1 ± 5.3 and 43.4 mg kg–1 ± 14.4 for alfaxalone and propofol, respectively, with no significant effect on Heinz body formation at any time point. Conclusions and Clinical relevanceHaematocrit decreased in both groups with recovery after 3 weeks. Repeated alfaxalone and propofol administration was not associated with marked haematological or serum biochemistry changes.

Glucose-6-Phosphate Dehydrogenase Deficiency and Neonatal Hyperbilirubinemia: Insights on Pathophysiology, Diagnosis, and Gene Variants in Disease Heterogeneity.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a prevalent condition worldwide and is caused by loss-of-function mutations in the G6PD gene. Individuals with deficiency are more susceptible to oxidative stress which leads to the classical, acute hemolytic anemia (favism). However, G6PD deficiency in newborn infants presents with an increased risk of hyperbilirubinemia, that may rapidly escalate to result in bilirubin induced neurologic dysfunction (BIND). Often with no overt signs of hemolysis, G6PD deficiency in the neonatal period appears to be different in the pathophysiology from favism. This review discusses and compares the mechanistic pathways involved in these two clinical presentations of this enzyme disorder. In contrast to the membrane disruption of red blood cells and Heinz bodies formation in favism, G6PD deficiency causing jaundice is perhaps attributed to the disruption of oxidant-antioxidant balance, impaired recycling of peroxiredoxin 2, thus affecting bilirubin clearance. Screening for G6PD deficiency and close monitoring of affected infants are important aspects in neonatal care to prevent kernicterus, a permanent and devastating neurological damage. WHO recommends screening for G6PD activity of all infants in countries with high prevalence of this deficiency. The traditional fluorescent spot test as a screening tool, although low in cost, misses a significant proportion of cases with moderate deficiency or the partially deficient, heterozygote females. Some newer and emerging laboratory tests and diagnostic methods will be discussed while developments in genomics and proteomics contribute to increasing studies that spatially profile genetic mutations within the protein structure that could predict their functional and structural effects. In this review, several known variants of G6PD are highlighted based on the location of the mutation and amino acid replacement. These could provide insights on why some variants may cause a higher degree of phenotypic severity compared to others. Further studies are needed to elucidate the predisposition of some variants toward certain clinical manifestations, particularly neonatal hyperbilirubinemia, and how some variants increase in severity when co-inherited with other blood- or bilirubin-related genetic disorders.

NEW REPORT OF KOLN HEMOGLOBIN FROM COUNTRYSIDE/INTERIOR OF SÃO PAULO

Hemoglobinopathies are considered a major public health problem in many countries, including Brazil, a fact explained by their frequency, genetic diversity and clinical importance. The unstable hemoglobin group is associated with congenital non-spherocytic hemolytic anemia due to instability of the hemoglobin molecule which can lead to the formation of characteristic inclusion bodies or Heinz bodies. Hb Köln beta 98(FG5) Val>Met is the result of the substitution of the amino acid valine for methionine at position 98 of the Beta chain of hemoglobin. Hemoglobin Köln reports are concentrated in Europe and in some parts of the Orient, such as northern Europe, among Jews, Czechoslovaks, Japanese, and also Koreans. A case was recently reported in India and, in Brazil, some cases have been known since 1993. This hemoglobinopathy is widely distributed, however, its frequency is extremely low when compared to the most frequent variants such as hemoglobin S (HbS) and C (HbC) in Brazil. Patients with Hb Köln usually present with mild hemolytic anemia characterized by reticulocytosis, splenomegaly and bilirubin, jaundice, and persistent dark urine excretion. In addition, some case reports describe Köln patients with priapism or persistent thrombocytopenia. We report a case of Köln's Hemoglobin in a 54-year-old female patient with hemolytic anemia under investigation. Osmotic instability tests were performed with a NaCl solution, hemoglobin electrophoresis in cellulose acetate at alkaline pH in which the presence of a band migrating between A 2 and S hemoglobins was observed, when the hemolysate was performed with 1% saponin, search for Heinz bodies with a 1:1 brilliant cresyl blue solution incubated at 37°C for 30 minutes, after which the bodies were identified in the blood smear. Upon suspicion of unstable hemoglobin, other standard laboratory procedures were performed, such as High Performance Liquid Chromatography (HPLC) with the Trinity®Premier Resolution Analytical Column equipment, in which a dosage of 3.18% HbA 2 and 0.16% HbF was observed. The results corroborate the electrophoretic and chromatographic profile suggestive of heterozygosity for Hb Köln. It is known today that there are 155 unstable hemoglobins identified worldwide and, of these, approximately 102 lead to hemolysis or abnormal oxygen affinity. In contrast the other variants do not show hematological abnormalities, but show instability in in vitro test. The phenotype of unstable variants of hemoglobin is diverse and dependent on the position of the mutation, which can lead to changes in the structure of the globin chain or of the hemoglobin molecule. Hb Köln is the most common of the unstable variants of hemoglobin and was first described by Pribilla in 1962. One of the main characteristics of unstable hemoglobins is the presence of Heinz bodies, which was curiously observed in this patient's red blood cells, corroborating data already described in the literature. Thus, a reliable detection of Hb Köln together with genetic counseling and prenatal diagnosis are essential to improve the well-being of patients.

Hematological and histological changes from ingestion of Deepwater Horizon crude oil in zebra finches (Taeniopygia guttata)

Exposure to crude oil during spill events causes a variety of pathologic effects in birds, including oxidative injury to erythrocytes, which is characterized in some species by the formation of Heinz bodies and subsequent anemia. However, not all species appear to develop Heinz bodies or anemia when exposed to oil, and there are limited controlled experiments that use both light and electron microscopy to evaluate structural changes within erythrocytes following oil exposure. In this study, we orally dosed zebra finches (Taeniopygia guttata) with 3.3 or 10 mL/kg of artificially weathered Deepwater Horizon crude oil or 10 mL/kg of peanut oil (vehicle control) daily for 15 days. We found that birds receiving the highest dosage experienced a significant increase in reticulocyte percentage, mean corpuscular hemoglobin concentration, and liver mass, as well as inflammation of the gastrointestinal tract and lymphocyte proliferation in the spleen. However, we found no evidence of Heinz body formation based on both light and transmission electron microscopy. Although there was a tendency for packed cell volume and hemoglobin to decrease in birds from the high dose group compared to control and low dose groups, the changes were not statistically significant. Our results indicate that additional experimental dosing studies are needed to understand factors (e.g., dose- and species-specific sensitivity) and confounding variables (e.g., dispersants) that contribute to the presence and severity of anemia resulting from oil exposure in birds.

The Impact of Glucose-6-Phosphate Dehydrogenase Deficiency on the Frequency of Vasoocclusive Crisis in Patients with Sickle Cell Anemia

Background and ObjectivesSickle cell anemia (SCA) and glucose-6-phosphate dehydrogenase (G6PD) deficiency are both hereditary diseases of the red blood cells that cause hemolysis. The impact of the interaction of both conditions on the clinical and laboratory presentations of the affected persons is sparse. This study, therefore, correlated G6PD activity with disease severity in persons with SCA by comparing disease severity in G6PD-deficient SCA persons with those with normal G6PD activity.MethodologyThis cross-sectional study was conducted in the department of Haematology and Blood Transfusion of the Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife, Nigeria. G6PD activity, SCA disease severity, and hematological parameters including reticulocyte counts and Heinz body estimation, bilirubin, and aspartate transaminase were estimated in 67 SCA persons. The results were compared between SCA persons with G6PD deficiency and those with normal enzyme activity.ResultsThe prevalence of G6PD deficiency was found to be 23.9%. The G6PD-deficient SCA patients included 4 (25.0%) males and 12 (75.0%) females. G6PD deficiency was significantly higher in females ( P = .047). There was no significant difference in disease severity scores between G6PD-deficient and G6PD-nondeficient SCA patients. However, G6PD-deficient persons reported significantly higher episodes of severe vasoocclusive crisis (VOC) per annum ( P = .048). The hematological and biochemical parameters were similar between G6PD-deficient and G6PD normal SCA persons except that the G6PD-deficient SCA persons have significantly higher reticulocyte response ( P = .001). There was no correlation between disease severity resulting from reduced G6PD activity and Heinz body formation in SCA persons in the steady state.ConclusionG6PD deficiency significantly contributes to recurrent painful vasoocclusive crisis in SCA persons in the steady state.

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Poisons affecting the blood

Blood circulates as a liquid containing cells and numerous chemicals; it functions to transport nutrients, chemicals, oxygen and waste products to and from cells, and is involved in defence and wound repair to tissues. Numerous toxic substances can disrupt the normal function of the blood through various mechanisms resulting in clinical signs of poisoning. Some of the substances that commonly cause adverse effects on the blood in companion animals are described. These include onions and related food plants which cause oxidative damage to red blood cells and the formation of Heinz bodies, and zinc which causes haemolytic anaemia. Haemoglobin is also subject to oxidative damage from paracetamol metabolites resulting in the formation of methaemoglobin which is non-functioning, resulting in tissue hypoxia. Disruption of the clotting cascade by anticoagulant rodenticides results in delayed-onset haemorrhage and the drug 5-fluorouracil disrupts the formation of blood cells causing bone marrow depression.

Poisons affecting the blood

Blood circulates as a liquid containing cells and numerous chemicals; it functions to transport nutrients, chemicals, oxygen and waste products to and from cells, and is involved in defence and wound repair to tissues. Numerous toxic substances can disrupt the normal function of the blood through various mechanisms resulting in clinical signs of poisoning. Some of the substances that commonly cause adverse effects on the blood in companion animals are described. These include onions and related food plants which cause oxidative damage to red blood cells and the formation of Heinz bodies, and zinc which causes haemolytic anaemia. Haemoglobin is also subject to oxidative damage from paracetamol metabolites resulting in the formation of methaemoglobin which is non-functioning, resulting in tissue hypoxia. Disruption of the clotting cascade by anticoagulant rodenticides results in delayed-onset haemorrhage and the drug 5-fluorouracil disrupts the formation of blood cells causing bone marrow depression.

Phenylhydrazine and its salts – calculated on phenylhydrazine. Documentation of proposed values of occupational exposure limits (OELs)

Phenylhydrazine and its salts – calculated on phenylhydrazine. Documentation of proposed values of occupational exposure limits (OELs)

Para-tertiary butyl catechol induces eryptosis in vitro via oxidative stress and hemoglobin leakage in human erythrocytes

Exposure of human population to industrial chemicals is believed as a significant contributing factor to the outgrowth of occupational diseases especially in developing countries due to improper safety measures and sanitary conditions. Para-tertiary butylcatechol (PTBC) widely employed in petrochemical, thermofax and phototypesetting industries, induces melanocytotoxicity and contact dermatitis leading to occupational leukoderma/vitiligo. Few vitiligo patients were reported for oxidative stress-induced hemolytic anemia and thrombocytopenia, however its impact on blood components is still not clear. Erythrocytes are the major cell population in circulation and play a prominent role in various diseases. In this work, the effect of PTBC on human erythrocytes is evaluated in vitro. PTBC induces oxidative stress-mediated eryptosis (erythrocyte death) causing detrimental changes such as depleted antioxidant levels, altered surface morphology, hemoglobin denaturation and heinz body formation. These findings validate that PTBC could induce toxic effects on human erythrocytes. Exposure of humans to toxic chemicals constitutes an important issue in various industries; one such issue is the exposure of PTBC at work place resulting in a spectrum of dermal complications. Therefore, it is imperative to appraise the long-term toxicities in order to further delineate the mechanisms of resultant disorders associated with PTBC and to establish the therapeutic interventions.

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH-Cytochrome B5 Reductase Gene.

BackgroundIn veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.ObjectivesTo analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.AnimalsThree Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.MethodsMethemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.ResultsMethemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).Conclusions and Clinical ImportanceThis dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.

Weathered MC252 crude oil-induced anemia and abnormal erythroid morphology in double-crested cormorants (Phalacrocorax auritus) with light microscopic and ultrastructural description of Heinz bodies

Injury assessment of birds following the Deepwater Horizon (DWH) oil spill in 2010 was part of the Natural Resource Damage Assessment. One reported effect was hemolytic anemia with the presence of Heinz bodies (HB) in birds, however, the role of route and magnitude of exposure to oil is unknown. The purpose of the present study was to determine if double-crested cormorants (Phalacocorax auritis; DCCO) exposed orally and dermally to artificially weathered crude oil would develop hemolytic anemia including HB and reticulocytosis. In the oral experiment, sub-adult, mixed-sex DCCOs were fed control (n = 8) or oil–injected fish with a daily target dose of 5 (n = 9) or 10 (n = 9) ml oil/kg for 21 days. Then, subadult control (n = 12) and treated (n = 13) cormorant groups of similar sex-ratio were dermally treated with approximately 13ml of water or weathered MC252 crude oil, respectively, every 3 days for 6 dosages approximating 20% surface coverage. Collected whole blood samples were analyzed by light (new methylene blue) and transmission electron microscopy. Both oral and dermal treatment with weathered DWH MC252 crude oil induced regenerative, but inadequately compensated, anemia due to hemolysis and hematochezia as indicated by decreased packed cell volume, relative increase in reticulocytes with lack of difference in corrected reticulocyte count, and morphologic evidence of oxidant damage at the ultrastructural level. Hemoglobin precipitation, HB formation, degenerate organelles, and systemic oxidant damage were documented. Heinz bodies were typically <2µm in length and smaller than in mammals. These oblong cytoplasmic inclusions were difficult to see upon routine blood smear evaluation and lacked the classic button appearance found in mammalian red blood cells. They could be found as light, homogeneous blue inclusions upon new methylene blue staining. Ultrastructurally, HB appeared as homogeneous, electron-dense structures within the cytosol and lacked membranous structure. Oxidant damage in avian red blood cells results in degenerate organelles and precipitated hemoglobin or HB with different morphology than that found in mammalian red blood cells. Ultrastructural evaluation is needed to definitively identify HB and damaged organelles to confirm oxidant damage. The best field technique based on the data in this study is assessment of PCV with storage of blood in glutaraldehyde for possible TEM analysis.

Reprint of: Overview of avian toxicity studies for the Deepwater Horizon Natural Resource Damage Assessment

The Oil Pollution Act of 1990 establishes liability for injuries to natural resources because of the release or threat of release of oil. Assessment of injury to natural resources resulting from an oil spill and development and implementation of a plan for the restoration, rehabilitation, replacement or acquisition of natural resources to compensate for those injuries is accomplished through the Natural Resource Damage Assessment (NRDA) process. The NRDA process began within a week of the Deepwater Horizon oil spill, which occurred on April 20, 2010. During the spill, more than 8500 dead and impaired birds representing at least 93 avian species were collected. In addition, there were more than 3500 birds observed to be visibly oiled. While information in the literature at the time helped to identify some of the effects of oil on birds, it was not sufficient to fully characterize the nature and extent of the injuries to the thousands of live oiled birds, or to quantify those injuries in terms of effects on bird viability. As a result, the US Fish and Wildlife Service proposed various assessment activities to inform NRDA injury determination and quantification analyses associated with the Deepwater Horizon oil spill, including avian toxicity studies. The goal of these studies was to evaluate the effects of oral exposure to 1–20ml of artificially weathered Mississippi Canyon 252 oil kg bw-1 day-1 from one to 28 days or one to five applications of oil to 20% of the bird's surface area. It was thought that these exposure levels would not result in immediate or short-term mortality but might result in physiological effects that ultimately could affect avian survival, reproduction and health. These studies included oral dosing studies, an external dosing study, metabolic and flight performance studies and field-based flight studies. Results of these studies indicated changes in hematologic endpoints including formation of Heinz bodies and changes in cell counts. There were also effects on multiple organ systems, cardiac function and oxidative status. External oiling affected flight patterns and time spent during flight tasks indicating that migration may be affected by short-term repeated exposure to oil. Feather damage also resulted in increased heat loss and energetic demands. The papers in this special issue indicate that the combined effects of oil toxicity and feather effects in avian species, even in the case of relatively light oiling, can significantly affect the overall health of birds.

Publisher's note

The Oil Pollution Act of 1990 establishes liability for injuries to natural resources because of the release or threat of release of oil. Assessment of injury to natural resources resulting from an oil spill and development and implementation of a plan for the restoration, rehabilitation, replacement or acquisition of natural resources to compensate for those injuries is accomplished through the Natural Resource Damage Assessment (NRDA) process. The NRDA process began within a week of the Deepwater Horizon oil spill, which occurred on April 20, 2010. During the spill, more than 8500 dead and impaired birds representing at least 93 avian species were collected. In addition, there were more than 3500 birds observed to be visibly oiled. While information in the literature at the time helped to identify some of the effects of oil on birds, it was not sufficient to fully characterize the nature and extent of the injuries to the thousands of live oiled birds, or to quantify those injuries in terms of effects on bird viability. As a result, the US Fish and Wildlife Service proposed various assessment activities to inform NRDA injury determination and quantification analyses associated with the Deepwater Horizon oil spill, including avian toxicity studies. The goal of these studies was to evaluate the effects of oral exposure to 1-20ml of artificially weathered Mississippi Canyon 252 oil kg bw-1 day-1 from one to 28 days or one to five applications of oil to 20% of the bird's surface area. It was thought that these exposure levels would not result in immediate or short-term mortality but might result in physiological effects that ultimately could affect avian survival, reproduction and health. These studies included oral dosing studies, an external dosing study, metabolic and flight performance studies and field-based flight studies. Results of these studies indicated changes in hematologic endpoints including formation of Heinz bodies and changes in cell counts. There were also effects on multiple organ systems, cardiac function and oxidative status. External oiling affected flight patterns and time spent during flight tasks indicating that migration may be affected by short-term repeated exposure to oil. Feather damage also resulted in increased heat loss and energetic demands. The papers in this special issue indicate that the combined effects of oil toxicity and feather effects in avian species, even in the case of relatively light oiling, can significantly affect the overall health of birds.

Interactions between peroxiredoxin 2, hemichrome and the erythrocyte membrane

Peroxiredoxin 2 (Prx2) is an abundant antioxidant protein in erythrocytes that protects against hemolytic anemia resulting from hemoglobin oxidation and Heinz body formation. A small fraction of Prx2 is bound to the cell membrane, but the mechanism and relevance of binding are not clear. We have investigated Prx2 interactions with the erythrocyte membrane and oxidized hemoglobin and whether these interactions are dependent on Prx2 redox state. Membrane binding of Prx2 in erythrocytes decreased when the cells were treated with H2O2, but studies with purified Prx2 and isolated ghosts showed that the interaction was independent of Prx2 redox state. Hemoglobin oxidation leads to the formation of hemichrome, a denatured form of the protein that binds to Band3 protein in the cell membrane as part of the senescence process and is a precursor of Heinz bodies. Hemichrome competed with Prx2 and decreased Prx2 binding to the membrane, potentially explaining the decreased binding in oxidant-exposed cells. The increased membrane binding of Prx2 seen with increasing intracellular calcium was less sensitive to H2O2 or hemichrome, suggesting an alternative mode of binding. Prx2 was also shown to exhibit chaperone-like activity by retarding the precipitation of pre-formed hemichrome. Our results suggest that Prx2, by restricting membrane binding of hemichrome, could impede Band3 clustering and exposure of senescence antigens. This mechanism, plus the observed chaperone activity for oxidized hemoglobin, may help protect against hemolytic anemia.

단기간 체중 감량이 RDW와 Heinz body 형성에 미치는 영향

단기간 체중 감량이 RDW와 Heinz body 형성에 미치는 영향

Skunk musk causes methemoglobin and Heinz body formation in vitro

A captive Red Panda developed a regenerative anemia with Heinz bodies after being sprayed by a skunk. A definite cause-and-effect relationship between skunk musk and oxidative erythrocyte damage has not been reported, but it was suspected in one reported case of a dog with Heinz body hemolytic anemia. The objective was to determine whether skunk musk induces oxidative HGB damage in vitro. Plasma and RBC were harvested from heparinized blood of 3 dogs, 3 cats, and a Red Panda. Skunk musk was solubilized in ethanol and mixed with plasma from each species to make stock solutions of 4% musk and 4% ethanol. Aliquots of RBC were resuspended in autologous stock solutions and solvent controls to yield musk concentrations of 0%, 0.04%, and 0.4% (by volume). Aliquots were incubated at 37°C for 4-72 hours and assessed for oxidative damage by visual inspection, optical absorbance spectroscopy, transmission electron microscopy, and light microscopy after Wright and vital New Methylene Blue staining. Dose-dependent brown color and absorption changes characteristic of methemoglobin were present by 4 hours and increased over 24 hours (Red Panda) and 72 hours (dog and cat). Similarly, there were time-dependent (all species) and dose-dependent (dog and cat) increases in the number of Heinz bodies, which were present by 4 hours and numerous by 24 hours. In vitro, skunk musk causes Heinz body and methemoglobin formation in canine, feline, and Red Panda RBC, supporting the clinical association between Heinz body hemolytic anemia and skunk spray exposure.

N,N-Dimethyl-p-toluidine, a Component in Dental Materials, Causes Hematologic Toxic and Carcinogenic Responses in Rodent Model Systems

Because of the potential for exposure to N,N-dimethyl-p-toluidine (DMPT) in medical devices and the lack of toxicity and carcinogenicity information available in the literature, the National Toxicology Program conducted toxicity and carcinogenicity studies of DMPT in male and female F344/N rats and B6C3F1/N mice. In these studies, a treatment-related macrocytic regenerative anemia characterized by increased levels of methemoglobin and Heinz body formation developed within a few weeks of DMPT exposure in rats and mice. DMPT induced nasal cavity, splenic, and liver toxicity in rats and mice at 3 months and 2 years. DMPT carcinogenic effects were seen in the liver of male and female rats and mice, the nasal cavity of male and female rats, and the lung and forestomach of female mice. In rodents, DMPT is distributed to many of the sites where toxic and carcinogenic effects occurred. DMPT-induced oxidative damage at these target sites may be one mechanism for the treatment-related lesions. Methemoglobinemia, as seen in these DMPT studies, is caused by oxidation of the heme moiety, and this end point served as an early alert for other target organ toxicities and carcinogenic responses that followed with longer term exposure.

Attenuation of oxidative hemolysis of human red blood cells by the natural phenolic compound, allylpyrocatechol

The protecting ability of the Piper betle leaves-derived phenol, allylpyrocatechol (APC) against AAPH-induced membrane damage of human red blood cells (RBCs) was investigated. Compared to control, AAPH (50 mM) treatment resulted in significant hemolysis (55%, p < 0.01), associated with increased malondialdehyde (MDA) (2.9-fold, p < 0.001) and methemoglobin (6.1-fold, p < 0.001) levels. The structural deformation due to membrane damage was confirmed from scanning electron microscopy (SEM) images and Heinz bodies formation, while the cell permeability was evident from the K+ efflux (28.7%, p < 0.05) and increased intracellular Na+ concentration (8%, p < 0.05). The membrane damage, due to the reduction of the cholesterol/phospholipids ratio and depletion (p < 0.001) of ATP, 2,3-DPG by ˜44–54% and Na+–K+ ATPase activity (43.7%), indicated loss of RBC functionality. The adverse effects of AAPH on all these biochemical parameters and the resultant oxidative hemolysis of RBCs were significantly reduced by pretreating the cells with APC (7 μM) or α-tocopherol (50 μM) for 1 h, prior to incubation with AAPH.