While red cells from individuals with ,8 thalassemias are characterized by evidence of elevated in vivo oxidation, it has not been possible to directly examine the relationship between excess a-hemoglobin chains and the observed oxidant damage. To investigate the oxidative effects of unpaired a-hemoglobin chains, purified a-hemoglobin chains were entrapped within normal erythrocytes. These "model" ,8-thalassemic cells generated significantly (P < 0.001 ) greater amounts of methemoglobin and intracellular hydrogen peroxide than did control cells. This resulted in significant time-dependent decreases in the protein concentrations and reduced thiol content of spectrin and ankyrin. These abnormalities correlated with the rate of a-hemoglobin chain autoxidation and appearance of membranebound globin. In addition, a-hemoglobin chain loading resulted in a direct decrease (38.5%) in catalase activity. In the absence of exogenous oxidants, membrane peroxidation and vitamin E levels were unaltered. However, when challenged with an external oxidant, lipid peroxidation and vitamin E oxidation were significantly (P < 0.001 ) enhanced in the a-hemoglobin chainloaded cells. Membrane bound heme and iron were also significantly elevated (P < 0.001 ) in the a-hemoglobin chain-loaded cells and lipid peroxidation could be partially inhibited by entrapment of an iron chelator. In contrast, chemical inhibition of cellular catalase activity enhanced the detrimental effects of entrapped a-hemoglobin chains. In summary, entrapment of purified a-hemoglobin chains within normal erythrocytes significantly enhanced cellular oxidant stress and resulted in pathological changes characteristic of thalassemic cells in vivo. This model provides a means by which the pathophysiological effects of excess a-hemoglobin chains can be examined. (J. Clin.

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A viable autosomal recessive mutation (named fch, or ferrochelatase deficiency) causing jaundice and anemia in mice arose in a mutagenesis experiment using ethylnitrosourea. Homozygotes (fch/fch) display a hemolytic anemia, photosensitivity, cholestasis, and severe hepatic dysfunction. Protoporphyrin is found at high concentration in erythrocytes, serum, and liver. Ferrochelatase activity in various tissues is 2.7-6.3% of normal. Heterozygotes (+/fch) are not anemic and have normal liver function; they are not sensitive to light exposure; ferrochelatase activity is 45-65% of normal. Southern blot analysis using a ferrochelatase cDNA probe reveals no gross deletion of the ferrochelatase gene. This is the first spontaneous form of erythropoietic protoporphyria in the house mouse. Despite the presence in the mouse of clinical and biochemical features unfrequent in the human, this mutation may represent a model for the human disease, especially in its severe form. (J. Clin.

In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta‐globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S‐Antilles‐D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2‐globin gene, each linked to the beta‐globin locus control region (LCR) were co‐introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD‐1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse‐human hybrids in addition to mouse hemoglobin. Adult SAD‐1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD‐1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta‐thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta‐thal/SAD‐1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.

Central to the pathophysiology of sickle cell disease are the vaso-occlusive events that lead to tissue damages and lifethreatening complications. Lungs are particularly vulnerable to vaso-occlusion because of their specific vasculature. We developed a mouse model of hypoxia/ reoxygenation lung injury closely mimicking the lung pathology of patients with sickle cell disease. This model involves the exposure of transgenic sickle cell (SAD) mice to hypoxia (8% oxygen) for 4, 10, and 46 hours followed by 2 hours of reoxygenation. Gene expression profiling of SAD lung tissue pointed to the specific induction of genes involved in the response to ischemic stress and microcir-