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 (α-FeOOH) iron oxide phase leads to lower levels of DNA damage