Med. Oncol. & Tumor Pharrnacother. Vol. 8. No. 4, pp. 229-233, 1991

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REVIEW IRON, FREE RADICALS

AND CANCER

PETER REIZENSTEIN Hematology Laboratory, Karoiinska Hospital, 10401 Stockholm, Sweden

(Received 29 April 1991 ; accepted in revised form 10 July 1991) Free radicals, intermediates in the tissue damage caused by radiation, are formed, inter alia, in interactions catalyzed by iron, which synergizes with radiation and some cytostatics (anthracyclins) in causing cell damage. Conversely, iron chelators can counteract cell damage. Similarly, antioxidants can slow atherogenesis, caused in part by oxidative stress and free radicals. Cell damage is also prevented by physiological defense systems like superoxide dismutase~ against endogenous free radicals formed by granulocytes, monocytes, etco Iron can thus induce free radicals which cause DNA double strand breaks and oncogene activation. This is suggested by four epidemiological studies suggesting a higher cancer risk in patients with larger iron stores than in those with small iron stores. In addition to its effect on carcinogenesis, iron can also maintain the growth of malignant cells as well as growth of pathogens. Breast cancer cells, for instance, display 5-15 times more transferrin receptors than normal breast tissue. Iron-carrying transferrin is in fact a growth factor. Hyposideremia in patients with cancer or infection is not a paraphenomenon but a functioning defense mechanism ('nutritional immunity'). If this immunity is broken by iron administration, relapses of diseases like tuberculosis, brucellosis, and malaria have been described. While iron-deficiency anemia should of course be diagnosed, treated and if possible prevented, there are good reasons to avoid over-utilization of medicamental iron.

Key words: Iron, Free radicals, Cancer. animals, as well as between SOD-levels and resistance to cytostatics in patients with leukemia. Normal granulocytes can generate free radicals, but not granulocytes from patients with chronic granulomatous disease. 3 In these patients, the capacity to generate the superoxide anion may be restored by interferon, IL-1, and GM-CSF. 4

INTRODUCTION

Free radicals~ Oxygen and hydroxyl radicals, inter alia, are toxic species, primary intermediates in the development of radiobiological damage. Free radicals.have unpaired electrons, but unlike ions they are not necessarily charged. Electrons may be unpaired in a particular shell even though their number may equal that of protons.t Oxygen can be reduced first to superoxide, then to hydrogen peroxide, and finally to hydroxyl radicals. Iron is essential to catalyze these reactions, and iron may also catalyze lipid peroxide formation. Iron can cause lipid peroxidation even in the form of ferritin, but not in that of lactoferrin. Lipid peroxidation can be terminated by, e.g., vitamin E or antioxidants. Superoxide dismutase (SOD) catalyzes dismutation of two superoxide radicals to water and O~. Proliferating lymphocytes display 10 times more S O D - m R N A than resting ones, and tumor necrosis factor-resistant cell lines 2-5 times more than sensitive ones. ~- A correlation has been found between constitutional SOD-levels and radioresistance in

Iron and free radicals. Free radicals are produced intracellularly in reactions catalyzed by iron 5 and possibly even by ferritin. 1 Superoxide can release iron from ferritin. Iron catalyzes the transformation of the non-toxic superoxide radical to toxic hydroxyl radicals. 6 Autooxidation of liposomes by Fe z+ can be reduced by polyamines bound to the liposome surface, changing a one-electron pathway generating superoxide to a two-electron pathway generating peroxide. Synergism between iron and radiation. Iron can synergize with radiation and radiomimetics in causing cell damage, and iron chelators seem to be able to prevent cell damage; for instance, in preventing adriamycin induction of iron-dependent hydroxyl 229

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radicals and subsequent D N A damage. (~ Increased iron content of cells increases radio sensitivity. 7 The cardiac toxicity of doxorubicin is blocked by iron chelators. Conversely, iron synergizes with anthracyclines in the generation of free radicals, s') Free radicals', cancer, rheumatoid arthritis and cardiovascular disease. Free radicals can, inter alia, activate oncogenes. Free iron and free radicals are present in the synovial fluid of patients with rheumatoid arthritis, m Free radicals produced by, inter alia, monocytes and endothelial cells can oxidize low density lipoproteins (LDL), which in turn attract more monocytes, reducing their interleukin-I and tumor necrosis factor production, altering their arachidonic acid metabolism, and inhibiting endothelial cell growth factor production. Oxidized LDL thus play a major role in atherogenesis, and circulating antibodies to oxidized LDL indicate the presence of these LDL in vivo in man. Lipofuscin, a lipid oxidation product, is found in both arteriosclerotic lesions and in patients treated with radiomimetic or cytostatic agents, t~ Lipofuscin accumulation can be inhibited by the chain-breaking free radical scavenger alpha tocopherol. Probucol, an antioxidant, can slow atherogenesis in rabbits. In man, serum lipid peroxides and glutathion peroxidase can reflect the risk of cardiovascular disease.12 It has also been suggested that catecholamine oxidation products can cause myocardial damage. Iron and cell growth. DNA-synthesis ~4 and ribonucleotide reductase 15 require iron. This is evidenced in the early S-phase (' of rapidly growing cells, not only by their expression of transferrin receptors ~6, but also by that of heavy-subunit ferritin (but not light-subunit ferritin) receptors. ~7 Since hydroxyurea inhibits ribonucleotide reductase, it synergizes with the iron chelator desferrioxamine. 6 Iron chelators also act as immunosuppressants by inhibiting T-cell proliferation (~, and they inhibit the S-phase in human lymphocyte proliferation. Iron is also required for 2-deoxy-nucleoside-5 triphosphate DNA-synthesis. Iron-deficient mice have more slowly growing tumors than those which are not deficient/' Leukemic children with a serum transferrin saturation > 36% had shorter survival than those with < 36%.as Hepatoma and infections are more common in patients with hemochromatosis. Similar findings have been made in BALB/c mice maintained on a normal iron diet, who developed colon carcinoma much more often than those on a diet low on iron. Similar findings were also made in other mouse

strains and other tumors. After administering a carcinogen, a low iron diet protected animals significantly against tumor development. Malignant cells have a high iron requirement and many transferrin receptors. The ferritin gene on chromosome 9 is partially homologous with the transferrin receptor region in the iron responsive element (IRE) region, where the IRE-binding protein switches from high to low affinity when iron is added. This switch is also seen in patients with hepatitis B, Fibs Ag (+) patients have a higher serum iron level and a higher iron saturation than Hbs Ag ( - ) subjects. In primary hepatic carcinoma, malignant cells show little stainable iron but normal hepatocytes show high levels of iron. Breast cancer cells, for instance, bind 5-15 times more transferrin than normal breast tissue, and similar differences have been found between highly malignant and low grade nonHodgkin lymphoma. ~" Autocrine mechanisms may occur in small cell lung cancer cells, which can synthesize transferrin. 6 That transferrin is not only a marker of rapid growth but in fact a growth factor is shown by the inhibition of transferrin receptor expression mediated by interferon. LsThus, iron chelators completely inhibit the proliferation of Daudi cells. ~ Similarly monoclonal antibodies against transferrin receptors can inhibit the growth of a T-cell line and of leukemia in mice. a h o n and infectious disease. Like cancer cells, microorganisms require iron. Lipid-soluble iron chelators have a strong anti-malarial effect, as well as an effect on the erythrocyte pool of free iron.15 Iron treatment of patients with anemia of chronic diseases has been shown to lead to relapses of tuberculosis, brucellosis, and malaria. Lactoferrin is an iron-binding protein acting as a bacteriostatic agent in milk, 6 and the unsaturated transferrin concentration is inversely correlated to the bacteriostatic activity of plasma. The bacteriostatic effect can be reversed if iron is added to saturate transferrin. The hyposideremia in patients with infections or cancer is therefore not a non-specific symptom, but a part of a defense reaction. ~9 Similarly, acidic isoferritin with heavy subunits is not only a tumor cell marker 2~ but also identical with leukemia inhibiting activity and. like lactoferrin, it inhibits growth of granulocyte-macrophage colonies, probably by interfering with cellular iron uptake. 6 Epidemiologicalstudies. Four epidemiological studies are reviewed by Stevens et al. ~ (1) In a H b A s + positive group of Chinese patients with ferritin > 300, primary hepatocellular carcino-

Iron, free radicals and cancer 231 ma was developed over a 2 - i 7 yr period twice as frequently as those with ferritin < 300. Ferritin was higher and transferrin was lower in subjects who subsequently died over a 10-year period and in those who developed cancer, than in control subjects. (2) In the so-called N H A N E S study, the transferrin saturation was 33.1% in those who developed cancer during a long follow-up period, vs 30.7% in those who did not. An exception is constituted by patients with cancer of the stomach, who exhibit lower serum ferritin levels than those who do not develop stomach cancer, possibly because of achlorhydria and reduced iron absorption. It is also claimed that oxygen-derived free oxygen radicals are implicated in the formation of duodenal ulcers. 2t (3) The lung cancer risk has been reported lower in women with low iron stores, i.e., anemia and a high total iron binding capacity, than in controls. Similarly, the lung-, colon-, bladder-, and esophagus cancer risk was higher in men with high iron stores. (4) Similarly again, the relative risk for cancer among almost 38,000 blood donors known to have chronic iron deficiency 2a,23"24 was only 0.79, showing a statistically significantly (P > .001) decreased cancer risk in blood donors. However, these findings were not confirmed in a fifth, Finnish study, so far published only in the form of a letter.

Overuse of fortification and tablets. Sweden probably has the world's highest, practically compulsory iron fortification of white flour (65 mg/kg), corresponding to 7 mg per inhabitant per day. z5 In addition, Sweden has sales of iron tablets 3--4 times higher than the United Kingdom, Germany, or France. 26 Nevertheless, a change of fortification to a more soluble and absorbable preparation, iron orthophosphate, has been recommended by a national expert group in Sweden. Much of this non-targeted fortification reaches groups other than, for instance, the 0.2% of men with iron deficiency or the 2.6-10% 7.28 of women in fertile age groups with large menstrual blood losses 2s at risk for iron deficiency, or preschool children with iron deficiency anemia. 29 Equal amounts of fortification iron are ingested by, for example, the 0.3% of Danish blood donors found to be non-diseased homozygotes for hemochromatosis, 3~ by patients with porphyria cutanea tarda, and by men with a high alcohol consumption. 3~ In fact, the number of people at risk of iron overload may be even greater, since the carrier rate of the autosomal recessive gene for hemochromatosis is 10-16% and the heterozygous frequency is 0.3-0.8%. v

There is evidence that alcoholic liver cell damage is mediated by iron catalyzed free radical lipid peroxidation, 32 and that radical scavenging enzymes are low in hepatitis. 33 Excess hepatic iron is found in 29% of alcoholics. 7

Proposals. The Danish National Board of Health recently recommended women to consult doctors before iron supplementation, and doctors to examine more thoroughly before prescribing iron. 34 A similar recommendation in Sweden has been proposed earlier, 35'36 particularly because overprescription of iron tablets is remarkably c o m m o n in the age groups at risk for cancer. At the age of 70, 7% of Swedish men and 12% of women have prescriptions for iron tablets. 3v However, on-going iron supplementation of the diet is needed in the minority of women in the fertile age group who are at risk of developing chronic iron deficiency because of large menstrual blood losses or frequent childbirth, in blood donors, and perhaps in some children. The question then is which preparation to use. While there is no consensus on this, something is to be said for heme iron, which has a more stable absorption than ferrous iron. Neither extreme inhibition of absorption nor extremely high absorption occur. A general use of heme iron could probably prevent the 17,000 cases of iron poisoning annually reported in the United States. 7 It is true that it has not yet been proved that iron is carcinogenic, nor that it facilitates the progress of tumors or infection, nor that it delays the diagnosis of anemia caused by gastrointestinal cancer, nor that it leads to the premature development of hemochromatosis or liver damage in at-risk groups. The important thing, however, is that avoidable or unnecessary iron supplementation has not been proved to be harmless in these respects. It seems well worth discussing now whether the time has come to restrict iron fortification, or at least make non-fortified white bread available. Overprescription of iron tablets to patients who do not have iron deficiency should be restricted, as required by the Danish National Board of Health, and supplementation targeted to a few children, some women, and blood donors as suggested earlier. Since both the risk of iron intoxication and that of free radical production is limited to the administration of ionizable iron, the possibility should be examined of giving supplementation in the form of heme iron, which does not increase the free iron concentration in the intestinal lumen, and which can not be absorbed in excess. In Sweden, groups at risk of developing chronic iron deficiency, such as women

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with constitutional excessive menstrual blood loss, could receive side-effect-free iron s u p p l e m e n t a t i o n free of charge. Swedish law provides for the free supply of substances of which patients have a chronic lack, such as vitamin B12 in pernicious anemia, insulin in diabetes, or cortisone in A d d i s o n s disease. The same should be true for iron in the few patients with a recurring, constitutional deficiency.

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