Pathophysiology of iron overload-induced renal injury and dysfunction: Roles of renal oxidative stress and systemic inflammatory mediators

Iron-overload has been recognized as a risk factor for organ dysfunction and damage resulting in diseases such as liver and heart disease, diabetes mellitus, and neurodegenerative diseases. This study investigated renal function and some systemic inflammatory indices in iron-overloaded male Wistar rats.Thirty animals were equally distributed into 3groups and treated daily i.p. with either normal saline (0.2 ml; control), iron (as ferrous sulphate) (15 mg/kg) or iron (30 mg/kg) for 21days respectively. Post-treatment, blood samples were obtained from each animal by cardiac puncture after light anaesthesia into plain sample bottles. Iron, ferritin, transferrin, creatinine, urea, albumin, total protein, interleukin-6 (IL-6), prostaglandins-E2 and tumor necrosis factor-α (TNF-α) were analysed in serum. Kidney homogenates were obtained per group and analysed for superoxide dismutase (SOD), total antioxidant capacity (TAC), reduced glutathione (GSH), lipid peroxidation (MDA) and nitric oxide (NO). Kidney histology was evaluated per group using both Haematoxylin and Eosin and periodic acid Schiff stains.Iron-overload caused a graded increase (p < 0.05) in serum iron, ferritin, transferrin, creatinine, urea, IL-6, TNF-α, TAC, MDA and NO levels as well as a reduction in albumin levels, renal SOD and GSH in groups 2 (iron 15 mg/kg) and 3 (iron 30 mg/kg) respectively compared to control. Histological evaluation of the kidney showed structural and tubular aberrations consistent with renal damage via inflammatory processes in iron overloaded rats.Our present study suggests that iron-overloading causes renal dysfunction by triggering the evolution of several inflammatory mediators which lead to a cascade of systemic and renal inflammatory processes that alter renal structure and function.     

Deferasirox and deferiprone remove cardiac iron in the iron-overloaded gerbil

Introduction: Deferasirox effectively controls liver iron concentration; however, little is known regarding its ability to remove stored cardiac iron. Deferiprone seems to have increased cardiac efficacy compared with traditional deferoxamine therapy. Therefore, the relative efficacy of deferasirox and deferiprone were compared in removing cardiac iron from iron-loaded gerbils. METHODS: Twenty-nine 8- to 10-week-old female gerbils underwent 10 weekly iron dextran injections of 200 mg/kg/week. Prechelation iron levels were assessed in 5 animals, and the remainder received deferasirox 100 mg/kg/D po QD (n = 8), deferiprone 375 mg/kg/D po divided TID (n = 8), or sham chelation (n = 8), 5 days/week for 12 weeks. RESULTS: Deferasirox reduced cardiac iron content 20.5%. No changes occurred in cardiac weight, myocyte hypertrophy, fibrosis, or weight-to-dry weight ratio. Deferasirox treatment reduced liver iron content 51%. Deferiprone produced comparable reductions in cardiac iron content (18.6% reduction). Deferiprone-treated hearts had greater mass (16.5% increase) and increased myocyte hypertrophy. Deferiprone decreased liver iron content 24.9% but was associated with an increase in liver weight and water content. CONCLUSION: Deferasirox and deferiprone were equally effective in removing stored cardiac iron in a gerbil animal model, but deferasirox removed more hepatic iron for a given cardiac iron burden.     

Effect of Iron-Chelator Deferiprone on the In Vitro Growth of Staphylococci

The standard iron-chelator deferoxamine is known to prevent the growth of coagulase-negative staphylococci (CoNS) which are major pathogens in iron-overloaded patients. However, we found that deferoxamine rather promotes the growth of coagulase-positive Staphylococcus aureus. Accordingly, we tested whether deferiprone, a new clinically-available iron-chelator, can prevent the growth of S. aureus strains as well as CoNS. Deferiprone did not at least promote the growth of all S. aureus strains (n=26) and CoNS (n=27) at relatively low doses; moreover, it could significantly inhibit the growth of all staphylococci on non-transferrin-bound-iron and the growth of all CoNS on transferrin-bound iron at relatively high doses. At the same doses, it did not at least promote the growth of all S. aureus strains on transferrin-bound-iron. These findings indicate that deferiprone can be useful to prevent staphylococcal infections, as well as to improve iron overload, in iron-overloaded patients.     

Transferrin receptors and selective iron deposition in pancreatic B cells of iron-overloaded rats.

Iron overload was produced in Wistar rats by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe(3+)-NTA) for one to six months. Pancreatic tissues from these iron-overloaded rats and untreated controls were examined for insulin (for B cells), glucagon (for A cells), transferrin receptor (TfR), transferrin (Tf) and ferritin (Ft) using immunohistochemical methods, and for iron by histochemical Berlin blue staining. In the islets of iron-overloaded rats, increased Ft staining appeared prior to deposition of Berlin blue-stainable iron, and the staining intensity of Ft and iron was stronger in B cells than in A cells. In the islets of untreated control rats, the staining intensity of TfR was stronger in B cells than in A cells. TfR staining of the islets was weaker in iron-overloaded rats than in the controls. These findings suggest that 1) iron uptake by islet cells in vivo is regulated and mediated by TfR, 2) intracytoplasmic Ft transforms into stainable iron in iron-overloaded rats, and 3) predominance of TfR expression in B cells may result in selective deposition of iron and predispose B cells to damage and diabetes mellitus in iron-overloaded rats.     

Failure of deferoxamine to improve iron overload in chronic hemodialysis patients

We prospectively studied the in vivo dialytic clearance of iron after deferoxamine (DFO) administration in four stable iron-overloaded chronic hemodialysis patients by quantifying iron concentration in blood entering and leaving the dialyzer and in dialysate after infusions of DFO. No significant arteriovenous iron differences were demonstrated. The mean ratio of venous to arterial iron approached identity at 1.005. All dialysate concentrates used contained large amounts of iron (300-610 micrograms/dl). No changes in efferent versus afferent dialysate iron concentration could be demonstrated. We conclude (a) iron removal during dialysis with DFO was not demonstrated; (b) the dialysate concentrate tested contained large amounts of iron; (c) in view of potentially significant toxicity, and lack of demonstrable therapeutic benefit, caution in prescribing DFO chelation therapy for iron overload is recommended.     

Transferrin Receptors and Selective Iron Deposition in Pancreatic B Cells of Iron-overloaded Rats

Iron overload was produced in Wistar rats by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe³⁺-NTA) for one to six months. Pancreatic tissues from these iron-overloaded rats and untreated controls were examined for insulin (for B cells), glucagon (for A cells), transferrin receptor (TfR), transferrin (Tf) and ferritin (Ft) using immunohistochemical methods, and for iron by histochemical Berlin blue staining. In the islets of iron-overloaded rats, increased Ft staining appeared prior to deposition of Berlin blue-stainable iron, and the staining intensity of Ft and iron was stronger in B cells than in A cells. In the islets of untreated control rats, the staining intensity of TfR was stronger in B cells than in A cells. TfR staining of the islets was weaker in iron-overloaded rats than in the controls. These findings suggest that 1) iron uptake by islet cells in vivo is regulated and mediated by TfR, 2) intracytoplasmic Ft transforms into stainable iron in iron-overloaded rats, and 3) predominance of TfR expression in B cells may result in selective deposition of iron and predispose B cells to damage and diabetes mellitus in iron-overloaded rats. Acta Pathol Jpn 41: 647–652, 1991.     

Ageing exacerbates the cardiotoxicity of hydrogen peroxide through the Fenton reaction in rats

Reactive oxygen species (ROS) are involved in the post-ischemic reperfusion syndrome of the myocardium. Moreover, ageing is associated with an increased cardiac sensitivity to both ischemia and reperfusion. The aim of the present study was to determine whether the lower tolerance of aged hearts to reperfusion could be due to an increased sensitivity to the ROS that are produced during the early phase of reperfusion. For this purpose isolated perfused hearts from adult (4 months) and aged (24 months) rats were perfused with a buffer containing 150 microM of hydrogen peroxide (H(2)O(2)) in presence or absence of deferoxamine mesylate (150 microM), an iron chelator. H(2)O(2) perfusion was continued until left ventricular developed pressure had decreased up to 20% of its initial value. Ageing led to a significant reduction of the duration of the H(2)O(2) perfusion required for inducing a 80% functional alteration. Although deferoxamine did not affect this parameter in adult rats, it significantly increased the duration of H(2)O(2)-perfusion in senescent hearts (control: 14.0+/-0.9 min vs. deferoxamine: 18.1+/-1.0, P<0.05). Similarly, ageing aggravated cardiac contracture induced by H(2)O(2)-perfusion. Again, deferoxamine, which had no effect on this parameter in young adult hearts, significantly reduced the contracture of senescent rat hearts. To conclude, our data clearly show that ageing is associated with an increased sensitivity of the myocardium to hydrogen peroxide, which is partly reversed by iron chelation. These results suggest that the iron-catalyzed Fenton reaction producing hydroxyl radicals might be greater in hearts from senescent rats than in hearts from young adults.     

Ventricular morphology and pumping ability of exercised spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) and two strains of normotensive Wistar rats were subjected to a 5 day/wk swimming program to determine whether the heart of the SHR could respond to an additional stimulus to cardiac growth. Swimming was tolerated well by all rats. Although body weight of the exercised groups was not significantly reduced, both the right and left ventricular weights of all exercised groups were increased. Left ventricular circumference and chamber volume were increased without a change in free wall thickness in all exercised groups. Ventricular performance was assessed by peak cardiac output and stroke volume attained during rapid intravenous volume loading, both before and after autonomic inhibition. After combined cholinergic and beta-adrenergic inhibition, all exercised rats had slower heart rates and higher peak stroke volume than respectively sedentary controls. Thus, exercised SHR had the same alterations in cardiac mass and performance as exercised normotensive rats. Despite the initial presence of left ventricular hypertrophy, the SHR responded appropriately to an additional stimulus for adaptive cardiac growth.     

实验性心肌肥厚大鼠血浆与心肌儿茶酚胺含量变化及黄芪的影响

结扎大鼠腹主动脉形成实验性心肌肥厚，对动物血浆及左心室组织中肾上腺素、去甲肾上腺素含量与心肌肥厚程度，以及黄芪注射液和卡托普利抗肥厚作用之间的相关性进行研究。手术4周后开始连续给药8周。以动物的心脏重量衡量心肌肥厚程度，用HPLC法测定血浆及左心室组织中肾上腺素和去甲肾上腺素含量，以SPSS统计软件对二者的相关性进行分析。结果显示模型组动物肾上腺素在血浆和心室组织中的含量均比对照组升高，去甲肾上腺素的血浆浓度比对照组升高，而其左心室组织中含量则低于对照组。黄芪注射液和卡托普利有不同程度的抗心肌肥厚作用，并可部分纠正肾上腺素和去甲肾上腺素含量的异常变化。相关性分析结果显示，心肌肥厚程度（全心重量、左心室重量）与左心室组织中去甲肾上腺素含量呈显著负相关性（P＜0．01）。上述结果提示：药物改变体内儿茶酚胺类物质含量可在一定程度上反映药物对心肌肥厚程度的影响。     

参松养心胶囊对糖尿病大鼠心室电生理特性及结构功能变化的影响

 目的：探讨参松养心胶囊（SSYX）对糖尿病（DM）大鼠心室电生理特性与结构功能变化的影响。方法：雄性SD大鼠45只，随机分为对照组（CTL组，n=15）、DM组（n=15）和SSYX组（n=15）。DM组和SSYX组采用一次性腹腔注射链脲佐菌素（60 mg/kg）制备糖尿病模型，CTL组则给予腹腔注射生理盐水（1 mL/kg），72 h后检测血糖评价造模结果，将造模成功的动物纳入本研究。SSYX组连续6周给予SSYX灌胃（1 g·kg-1·d-1）治疗，DM组和CTL组则给予生理盐水灌胃（2 mL·kg-1·d-1）。动物完成药物治疗后，采用超声心动图检测心功能并记录II导联体表心电图；采用放射免疫法检测血浆的内皮素1（ET-1）水平；随后在整体心脏Langendorff灌流条件下行离体电生理研究，分别记录和测量左室前游离壁（LAF）心外膜单相动作电位（MAP）和有效不应期（VERP），同时给予Burst快速电刺激用以进行室性心律失常（VA）的诱发；采用Masson染色对3组动物心肌纤维化程度进行评价。结果：与CTL组相比，DM组心功能下降（P<001），而QT间期、VERP、动作电位时程、VA诱发率、心肌纤维化程度和血浆ET-1水平均增加（均P<001）；与DM组相比，SSYX组的心功能得到改善（均P<001），且 QT间期、VERP、动作电位时程、VA诱发率、心肌纤维化程度和血浆ET-1水平均降低（均P<005）。结论：SSYX具有减轻DM大鼠心室电重构和结构重构及改善心功能的作用。     

Performance characteristics of three serum iron and total iron-binding capacity methods in acute iron overdose.

Accurate serum iron and total iron binding capacity (TIBC) measurements may be useful in acute iron overdoses. Two alumina column TIBC methods were found to measure increased TIBC when free iron was present. A homogeneous TIBC method gave consistent results until iron concentrations exceeded 500 micrograms/dL (90 mumol/L), when it began to underestimate the TIBC. Serious iron overdoses require chelation therapy with deferoxamine. Iron recovery was reduced by up to 50% for all 3 methods with clinically achievable concentrations of deferoxamine 8,400 micrograms/dL (150 mumol/L). TIBC measurements by both alumina column methods were reduced by deferoxamine in the presence of free iron and unaffected when the iron concentration was less than the TIBC. The homogeneous TIBC method yielded falsely elevated results in the presence of free deferoxamine. Procedures that measure TIBC by addition of excess ferric iron followed by alumina adsorption are not suitable for monitoring TIBC in acute iron overdose. The homogeneous TIBC assay can be used in acute iron overdose but underestimates TIBC when iron concentrations exceed 500 micrograms/dL (90 mumol/L). None of the methods examined are useful for measuring iron or TIBC in the presence of deferoxamine.     

Development of SHR hypertension and cardiac hypertrophy during prolonged beta blockade.

Spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were treated with beta-adrenergic receptor inhibiting drugs (either propranolol or timolol) from conception until 12 weeks of age to determine if this therapy would alter the development of systemic hypertension or left ventricular hypertrophy. Therapy (propranolol or timolol, 500 mg/liter drinking water) was initiated with breeding parents and continued throughout the pregnancy, nursing, and postweaning periods. Although the heart rates of beta-adrenergic receptor inhibited WKY and SHR rats were consistently reduced with respect to their respective tap-water controls, this therapy did not alter body growth. Hemodynamic studies demonstrated reduced central venous pressure, cardiac index, and maximum acceleration of aortic flow in the beta-adrenergic inhibited rats. In spite of these findings, the arterial pressure of the treated rats and the degree of left ventricular hypertrophy of the SHR were unaltered by treatment. Thus, administration of the beta-adrenergic receptor blocking agents, propranolol or timolol, from conception through the developmental stage of SHR hypertension, failed to alter either the progressive rise in arterial pressure or the development of hypertensive vascular disease and left ventricular hypertrophy.     

抑制Rac1通过降低磷酸化p38MAPK提高1型糖尿病小鼠的心脏功能

目的:探讨抑制Rac1对1型糖尿病小鼠心肌细胞肥大、心脏功能的影响及其作用机制。方法:50只8周龄雄性C57小鼠随机分为对照组(control,n=10)、Rac1抑制剂NSC23766对照组(NSC,n=10)、1型糖尿病组(STZ,n=15)及NSC治疗组(STZ+NSC,n=15)。小鼠腹腔注射链脲佐菌素(STZ)建立1型糖尿病动物模型,血糖升高后给予小鼠腹腔注射NSC23766,实验于8周末结束,记录实验小鼠生存率、测量小鼠体重及左室重量并计算左室重量指数,运用超声心动图检测小鼠心脏功能,心肌组织进行HE染色结合图像分析软件定量心肌细胞大小,运用实时定量RT-PCR技术检测心肌组织心房利钠肽(ANP)、脑利尿肽(BNP)、β-肌球蛋白重链(β-MHC)mRNA表达,以Westernblotting定量心肌组织磷酸化p38丝裂素活化蛋白激酶(pho-p38MAPK)表达。结果:抑制Rac1后:(1)糖尿病小鼠生存率提高、糖尿病小鼠左室重量指数降低(P0.01)、心脏左室射血分数(EF)增加、左室短轴缩短率增加(FS)(P0.01);(2)心肌细胞大小明显降低(P0.01);(3)心肌肥大相关基因ANP、BNP、β-MHC表达明显降低(P0.01);(4)心肌组织pho-p38MAPK表达明显降低(P0.01)。结论:抑制Rac1活性显著改善1型糖尿病小鼠心脏功能、降低心肌细胞肥大,其机制可能与明显降低心肌组织磷酸化p38MAPK密切相关。     

去铁酮和去铁胺治疗珠蛋白生成障碍性贫血临床疗效及安全性的Meta分析

目的采用Meta分析对去铁酮＋去铁胺联合治疗重型珠蛋白生成障碍性贫血的临床疗效及安全性进行综合定量评价。方法制定原始文献的纳入标准、排除标准以及检索策略,检索Cochrane图书馆、PubMed、EMBASE、Ovid、Springer、中国生物医学文献光盘数据库、中国期刊全文数据库和维普期刊数据库,检索时间为1985年1月1日至2008年5月31日,获得去铁酮＋去铁胺联合治疗重型珠蛋白生成障碍性贫血的相关文献。采用Cochrane中心推荐的方法对文献质量进行评价。选取血清铁蛋白（SF）、肝组织铁浓度（LIC）、心肌铁含量和不良反应发生率作为主要测量指标,总铁排泄量和心功能作为次要测量指标。采用Review Manager4．3．2软件对满足纳入标准的RCT文献进行Meta分析。计量资料采用加权均数差（WMD）或标准化均数差（SMD）及其95％CI表示,计数资料采用RR及其95％CI表示。结果共检索到相关文献216篇,符合纳入标准的5篇RCT文献（n=229）进入Meta分析,其中4篇文献质量评价为B级,1篇为C级。去铁酮＋去铁胺联合治疗组（联合治疗组）与单用去铁胺治疗组（去铁胺组）比较：治疗12个月后sF下降程度：SMD=-0．04,95％CI：-0．48～0．41;LIC下降程度：SMD=-0．1,95％CI：-0．47—0．27;总铁排泄量：WMD=0．18,95％CI：0—0．37,两组差异均无统计学意义。左心室射血分数改善情况：WMD=3．40,95％CI：0．97～5．82,提示左心室射血分数联合治疗组高于去铁胺组。安全性分析：胃肠道不良反应发生率联合治疗组高于去铁胺组,RR=2．77,95％CI：1．41—5．42。联合治疗组关节不良反应发生率：RR=1．45,95％CI：0．59～3．52;中性粒细胞减少症发生率：RR=0．85,95％CI：0．27～2．68;注射部位不良反应发生率：RR=0．57,95％CI：0．20～1．65,与去铁胺组差异均无统计学意义。1篇文献采用MRIT2^＊测定心肌铁含量,结果显示联合治疗组心肌铁含量下降程度高于去铁胺组。结论联合治疗组的短期疗效及安全性与去铁胺组相当,去除心肌铁含量和改善心功能方面效果优于去铁胺组;联合治疗组最常见的不良反应为胃肠道反应。因纳入文献较少,临床应用本研究结果应谨慎,进一步结论仍有待大样本、多中心和高质量的RCT研究来验证。     

Cardiac function and morphology with aging in the spontaneously hypertensive rat.

To determine the effects of a chronic pressure load on cardiac function and morphology, spontaneously hypertensive rats (SHR) and two normotensive strains of Wistar rats (WKY and NWR) were studied under ether anesthesia at 13, 25, 52, and 90 wk of age. Although resting cardiac index of the SHR was comparable to that of WKY and NWR at all ages, the peak cardiac output and peak stroke volume per gram of left ventricle determined during a rapid intravenous infusion of Tyrode solution was markedly reduced in the SHR only at 90 wk of age. Autonomic inhibition did not alter the peak stroke volume attained, but reduced peak cardiac output at all ages in each of the strains. Absolute left ventricular dimensions in the SHR increased out of proportion to body growth, consistent with concentric hypertrophy. As peak pumping ability markedly declined from 52 to 90 wk of age in the SHR, the free wall of the left ventricle greatly thickened whereas the septum remained unchanged. At this time the right ventricle also hypertrophied. This disproportionate thickening of the walls of the left ventricle and the hypertrophy of the right ventricle were reflected in measurements of their fiber diameters. These alterations in ventricular architecture may contribute to the decrease in pumping ability observed in long-standing hypertension.     

Inhibition of caries in hamsters treated with staphylococcin 1580.

Littermate hamsters were infected orally with cariogenic Streptococcus mutans NS-50S and maintained on a high-sucrose diet to induce dental caries. Individual groups of animals were treated by single daily instillation of staphylococcin 1580 (100 micrograms) in the cheek pouches. Other groups were treated similarly with bacitracin (60 or 120 U) or saline. After 35 days on the caries test regimen, animals treated with staphylococcin 1580 had 49 or 60% less caries than the saline controls in two separate trials. Hamsters treated with 60 U of bacitracin had 40% less caries, while animals receiving 120 U of bacitracin had 58% less caries. Significant reductions in the oral and fecal levels of S. mutans NS-50S were also observed in the treated animals. In contrast to earlier, inconclusive experiments in which these agents were placed directly into the oral cavity, the inhibition of caries in this study is attributed to prolongation of the oral retention of these agents, with increased opportunity to interact with cariogenic microorganisms.     

山莨菪碱对缺血心肌的保护作用

本文观察山莨菪碱(654-2)对大鼠缺血心肌的保护作用。发现大鼠左冠状动脉主干结扎后早期腹腔注射654-2,可使结扎后6小时及21天时的梗塞范围显著缩小,并使21天时的左室心肌收缩性恢复得更好,而结扎后3小时缺血区心肌的超微结构改变得到显著改善。654-2对冠脉结扎与非冠脉结扎大鼠的直接血液动力学作用表现为平均动脉压、心输出量和左室心肌收缩性显著降低,提示654-2可以减少心肌氧耗量,从而对缺血心肌发生有利影响。     

Effects of iron overload on the immune system

Iron and its binding proteins have immunoregulatory properties, and shifting of immunoregulatory balances by iron excess or deficiency may produce severe, deleterious physiological effects. Effects of iron overload include decreased antibody-mediated and mitogen-stimulated phagocytosis by monocytes and macrophages, alterations in T-lymphocyte subsets, and modification of lymphocyte distribution in different compartments of the immune system. The importance of iron in regulating the expression of T-lymphocyte cell surface markers, influencing the expansion of different T-cell subsets, and affecting immune cell functions can be demonstrated in vitro and in vivo. The poor ability of lymphocytes to sequester excess iron in ferritin may help to explain the immune system abnormalities in iron-overloaded patients. Iron overload as seen in hereditary hemochromatosis patients enhances suppressor T-cell (CD8) numbers and activity, decreases the proliferative capacity, numbers, and activity of helper T cells (CD4) with increases in CD8/CD4 ratios, impairs the generation of cytotoxic T cells, and alters immunoglobulin secretion when compared to treated hereditary hemochromatosis patients or controls. A correlation has recently been found between low CD8+ lymphocyte numbers, liver damage associated with HCV positivity, and severity of iron overload in beta-thalassemia major patients. Iron overload, with its associated increases of serum iron levels and transferrin saturation, may cause a poor response to interferon therapy. Iron overload with hyperferremia is associated with suppressed functions of the complement system (classic or alternative types). High plasma ferritin content in patients with chronic, diffuse diseases of the liver (cirrhosis, chronic hepatitis), beta-thalassemia major, dyserythropoiesis, and hereditary hemochromatosis may induce the development of anti-ferritin antibodies with the production of circulating immune complexes. Increased body stores of iron in various clinical situations may tip the immunoregulatory balance unfavorably to allow increased growth rates of cancer cells and infectious organisms, and complicate the clinical management of preexisting acute and chronic diseases.     

Effect of iron salts, haemosiderins, and chelating agents on the lymphocytes of a thalassaemia patient without chelation therapy as measured in the comet assay

Impairment of haemoglobin synthesis occurs in the genetic diseases known as thalassaemia. The consequent chronic anaemia leads to increased dietary iron absorption which results in iron overload. Treatment through regular blood transfusions increases oxygen capacity, but also adds iron from haemoglobin. An essential treatment, in parallel with transfusions, is the use of chelating agents to remove the excess iron. Thalassaemia patients are particularly at risk of free radical damage. Human lymphocytes from normal individuals can be investigated in vitro as a model system in the presence of free radicals in the Comet assay. This assay measures DNA damage, particularly DNA strand breakage. We examined cells from an Australian thalassaemic patient (sickle/beta thal double heterozygote-sickle phenotype) who had not yet received chelation therapy to determine if the cells were more sensitive to simulated iron overload and to haemosiderins. Lymphocytes from the patient were received as frozen samples after 28 h on dry ice and then placed in liquid nitrogen. Normal lymphocytes frozen under the same conditions and normal nonfrozen lymphocytes were compared. The lymphocytes from a normal female did not respond in vitro to ferric chloride (FeCl(3)) or haemosiderin but did to ferrous chloride (FeCl(2)) and ferrous sulphate (FeSO(4)). Deferoxamine appeared to reduce the response to FeCl(2) and FeSO(4) but deferiprone did not. When the lymphocytes from the nonchelated patient were treated with FeSO(4) and hydrogen peroxide, deferoxamine and deferiprone both reduced the response. Over the same dose range of iron salt (FeSO(4)), the lymphocytes from the thalassaemic patient were more sensitive, with much higher background levels of damage and induced damage. When deferiprone and deferoxamine were compared over a nontoxic range, deferiprone appeared to produce a greater reduction of damage in lymphocytes of the thalassaemia patient. Ferritin iron appears to be more available than haemosiderin iron in reactions leading to DNA damage. Haemosiderin containing higher amounts of the goethite-like (alpha-FeOOH) iron oxide phase leads to lower levels of DNA damage.