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Essential Metalloelement Chelates Facilitate Repair of Radiation Injury

Treatment with essential metalloelement (Cu, Fe, Mn, and Zn) chelates or combinations of them before and/or after radiation injury is a useful approach to overcoming radiation injury. No other agents are known to increase survival when they are used to treat after irradiation, in a radiorecovery treatment paradigm. These chelates may be useful in facilitating de novo syntheses of essential metalloelement-dependent enzymes required to repair radiation injury. Reports of radioprotection, which involves treatment before irradiation, with calcium-channel blockers, acyl Melatonin homologs, and substituted anilines, which may serve as chelating agents after biochemical modification in vivo, as well as Curcumin, which is a chelating agent, have been included in this review. These inclusions are intended to suggest additional approaches to combination treatments that may be useful in facilitating radiation recovery. These approaches to radioprotection and radiorecovery offer promise in facilitating recovery from radiation-induced injury experienced by patients undergoing radiotherapy for neoplastic disease and by individuals who experience environmental, occupational, or accidental exposure to ultraviolet, x-ray, or γ-ray radiation. Since there are no existing treatments of radiation-injury intended to facilitate tissue repair, studies of essential metalloelement chelates and combinations of them, as well as combinations of them with existing organic radioprotectants, seem worthwhile.

Transcription regulating proteins Endopeptidases required for repair, maintenance, and function of extracellular connective tissue components and wound repair Metalloelement transcription factor, a Zn-finger regulatory protein required for cellular response in metalloelement uptake Essential Metalloelement Metabolism Ingested foods and beverages contain these essential metalloelements in chelated forms, which may yield other chelates as a result of ligand exchange in the digest or following absorption [4]. However, some of the absorbed chelates may exist in the digest and in blood as the original chelate or a more stable ternary chelate. Absorbed metalloelement chelates undergo systemic circulation to all tissues and utilization by all cells following ligand exchange with small molecular mass ligands, apoproteins, and apoenzymes to form metalloproteins and metalloenzymes in de novo syntheses. These metalloelements are stored in the appropriate tissues as MTs or Fe-Ferritin, or they are excreted in the event tissue needs have been met and stores replenished [4]. There are no inducible excessive storage diseases known in normal individuals. Hovever, there must be upper limits dictated by physiological requirements.
Stored essential metalloelements are released as chelates via ligand exchange to meet normal metabolic needs. This homeostatic release of relatively small amounts of essential metalloelement chelates meets normal physiologic requirements. Release of larger quantifies in a pronounced mobilization of these metalloelements is a feature of interleukin-1 (IL-1) and other cytokine mediated acute and chronic responses to many disease states [4 and cited refs].
It is likely that the magnitude of these responses is adequate in normal well-nourished individuals and allows them to overcome their disease. However, it is now generally recognized that dietary intakes of Cu, Fe, Zn [37,38], and most likely Mn are less than recommended daily intakes for the U.S. population and may be no better for individuals living in other developed countries. Individuals who have depleted stores of these essential metalloelements, such as chronic disease patients, may overcome pathology associated with their disease state, which always involves inflammation, more slowly or not at all. The degree of radiation injury and the nutritional state of health of an individual may determine whether or not an individual will be able to overcome metalloelement-dependent repairable radiation injury. Treatment with essential metalloelement chelates either before and/or after radiation injury may support these essential metalloelement-dependent responses or facilitate recovery regardless of the state of nutriture.

Chemical Consequences of Ionizing Radiation
Since water represents 70% of the chemical composition of the adult body (90% of the infant body), its chemical transformation by ionizing radiation merits serious consideration with regard to chemical consequences of ionizing radiation. Ionizing radiolysis of water is well understood and produces very. reactive aquated electrons, monoatomic hydrogen atoms, hydroxyl radical, hydrogen peroxide, and protonated water as well as superoxide (O2-) and hydroperoxyl radical in the presence of oxygen. Hydroperoxyl radical, hydroxyl radical, monoatomic hydrogen, and aquated electrons have very short half-lives of the order of milliseconds and consequently react rapidly with cellular components in reduction, oxidation, initiation, insertion, propagation, and addition reactions causing loss of function and the need for biochemical replacement and/or repair. Ionizing radiation can also impart sufficient energy to all biochemicals to cause homolyltic bond breaking and produce all conceivable organic radicals in considering C-C, C-N, C-O, C-H, P-O, S-O, etcetera bond homolyses. These radicals will undergo the above listed radical reactions to cause further destruction and the need for replacement and/or repair. A third consequence of ionizing radiation is homolytic or heterolytic bond breaking of coordinate-covalent bonded metalloelements. These are the weakest bonds in biochemical molecules and potential sites of greatest damage, which may be most in need of replacement and/or repair since many repair enzymes are metalloelement-dependent.
Recognizing that loss of essential metalloelement-dependent enzyme activity may at least partially account for lethality of ionizing radiation and that Cu-, Fe-, Mn-, and Zn-dependent enzy.mes have roles in protecting against accumulation of Oas well as facilitating repair [39] may explain the radiation protection and radiation recovery" activities of Cu, Fe, Mn, and Zn compounds. It is suggeed that the IL-1 mediated redistributions of essential metalloelements have a general role in responding to radiation injury.. These redistributions may also account for subsequent de novo synthesis of metalloelement-dependent enz3'mes required for biochemical repair and replacement of cellular and extracellular cofiaponents needed for recover from radiolytic damage. De novo syntheses of metalloelement-dependent enzymes required for utilization of o.xTgen and prevention of O2accumulation as well as tissue repair processes including metalloelement-dependent DNA and RNA repair are key to the hypothesis that essential metalloelement chelates decrease and/or facilitate recovery from radiation-induced pathology. A widely held understanding is that O_accumulates in mammalian cellular and extracellular spaces due to the lack of normal concentrations of Cu and Zn-or Mndependent superoxide dismutases (SODs), inappropriate reduction of O_ leading to relatively large steady-state Oxidase which does synthesize 02has been recently demonstrated in the liver of 3 to 9 Gray (Gy) irradiated mice [40]. More reactive oxygen-radicals derived from 02include: singlet oxygen and hydrogen peroxide, produced as a result of acid-dependent self disproportionation or water-catalyzed dispt)portionation of O_-.
hydrox3'l radical, and hydroperoxyl radical [41,42]. Facilitated de now syntheses of Cu_Zn_,SODs and catalase and decreasing or preventing formation of these oxygen-radicals may account for some of the recovery from pathological changes associated with irradiation, which cause systemic inflammatory disease and immunoincompetency in the case of whole-body irradiation, and focal inflammatory., disease associated with local irradiation.
Radiation-Induced Tissue Injury Ionizing radiation is known to cause a variety of tissue injuries at all levels of biochemical systems. Acutely toxic radiation syndromes are well known in terms of doses of radiation and clinical courses of these syndromes [43]. Doses of radiation that are less than those that cause death in the short term as well as ultraviolet irradiation cause a variety of disease states depending upon the biochemical injury of the affected tissues. Consequences of ionizing and ultraviolet irradiation are a matter of current concern. Radiation risk of fatal malignancy associated with commonly performed coronary angiography was found to be around in 6,000 with a risk of fatal lung cancer of about in 8,000, an increase of 0.017% above the normal incidence for the general population [44]. Longer and more complicated procedures carry a greater risk consistent with the well known phenomenon of radiation-induced carcinogenesis [45]. Radiation-Induced skin injury, is also an unwanted consequence of this procedure and more so for all ex vivo irradiations of neoplastic tumors [44].
Adult CF male rats x-irradiated with 0.015 centiGy see 1 day 1 for 7 days followed by 7 days rest for either 3 months or 6 months showed duration related patchy skin hyalinization, which became completely hyalinized during the succeeding 6 months without irradiation [46]. The concentration of Zn in skin of these rats remained normal in both irradiated groups but by the end of yr it had significantly (P < 0.001) decreased to 13 lag grn " of tissue from 19 lag grn". Skin Fe was elevated in both irradiated groups and the elevation increased from 100 lag gm " of tissue with 3 mo irradiation to 140 lag grn after 6 mo irradiation versus 68 gm "1 for control skin, There was a hmher relative increase in skin Fe yr after irradiation to 129 tg grn "1 of tissue compared to 65 lag gm for control skin (P< 0.001). It was concluded that chronic exposure to low dose x-irradiation possibly causes a redistribution of Zn and Fe with concomitant hyalinization of collagen. The observed skin collagen pathology and altered essential metalloelement contents were interpreted as results of chronic inflammation due to chronic irradiation [46].
Recent studies of pregnant women who resided in a heavily exposed area of the Republic of Belarus following the Chemobyl catastrophe appear to be at risk for development of toxemia, renal insufficiency, and anemia. Neonates born in heavily contaminated areas were found to be at risk for development of anemia, congemtal malformations, and perinatal death. Decreased concentrations of erythrocyte Cu and Zn were documented for these neonates. Diminished white blood cell, T-helper cell, and T4 cell counts were found for neonates from areas of heavy exposure [47].
Radiation Protection and Recovery with Copper Chelates.
All radiation protection and radiation recovery doses of Cu(II)2(3,5-DIPS)4 Cu(II)_(3,5-DIPS)4 are non-toxic and ranged from 1/5 to 1/100 of an acutely toxic dose (LDs0/7) for male or female mice [see 48]. The LD50/7, acute toxicity, of Cu(II)2(3,5-DIPS) in female mice and rats, 261+36 tmol kg , was much higher than observed for male mice and rats, 91+ 13 prnol kg". Acutely lethal doses of Cu(II)2(3,5-DIPS)4 produce marked CNS depression due to loss of parasympathetic regulation of cardiac and pulmonary function. Smaller doses produce short duration hypnotic or sedative eftLcts. The small doses of Cu(II)2(3,5-DIPS4 exhibiting radioprotectant and radiorecovery activity do not produce any of these CNS depressant effects. Of perhaps still greater interest with regard to many studies of supposed "Cu-mediated toxicity" as having a role in an "oxidative etiology", suggested to account for pathology of many disease states, is the recent report pointing out that these studies were seriously flawed [51]. The same increase in 532 nm absorbance measured following the addition of thiobarbituric acid to disease-related biological systems to which ionically bonded forms of Cu had been added to "catalyze oxidations", was observed following addition of ionieally bonded Cu to aqueous solutions of only thiobarbituric acid. The linear increase in this 532 nm absorbance was related to the increase in Cu-thiobarbiturate chelate(s)formation.
Initial studies involving treatment of LD00/50 irradiated mice ffter irradiation employed a dose of 80 prnol Cu(II)2(3,5-DIPS)4 kg "1 which was found to be effective in increasing survival when given before irradiation. However this dose given nfter irradiation caused acute lethality, all of these treated mice died before the vehicle-treated mice (non-published). The rationale for this observation is that treatment with this large dose of Cu(II)2(3,5-DIPS)4 niter irradiation interfered with de novo synthesis of other essential metalloelement-dependent enzymes required for repair of radiation injury and decreased survival. That is, only a fraction of the dose given before radiation remains when radiation-injury occurs and this fraction is useful in overcoming radiation-injury and increasing survival. Giving this large dose of Cu(II)2(3,5-DIPS)4 ffter irradiation may interfere with the synthesis of other metalloelement-dependent enzymes by enabling incorporation of the incorrect metalloelement, in this case Cu, when the larger dose is given ffter radiation injury. This possibility also offers a rationale for historical observations that large doses of Cu, Fe, Mn, and Zn compounds cause "radiosensitization", reducing survival and increasing radiation-induced mortality. Large doses of aminothiol radioprotectants that are effective in increasing survival of lethally irradiated mice when given before irradiation are also lethal when given ter irradiation.  20, 40, 60, 80, 100, or 120 prnol kg l given sc before irradiation revealed that 80 or 120 prnol kgproduced significant increases in survival, 68% (P 0.019) or 92% (P 0.0008), respectively, compared to vehicletreated control mice [52].
Copper(II)(3,5-DIPS)4 did not prevent hematopoietic and immunologic damage to irradiated mice, since 98-99% of myeloid progenitor cells were destroyed and immune reactivity was ablated. However, Cu(II),_(3,5-DIPS) did accelerate cellular repopulation of bone manw and spleen following irradiation. Recovery of hematopoietic activity was accelerated whether mice were treated with Cu(II)2(3,5-DIPS) 3 hr before or 3 hr after irradiation and treatment increased levels of multipotential progenitor cells, which serve as a sotu'ce of both myeloid and lymphoid progenitor cells, to account for recovered responses to T and B cell mitogens and antibody responses to a T-dependent antigen [see 48]. Radiorecovery and/or radioprotectant activity of Cu-containing compounds or complexes should be recognized as possibly facilitating activation of Cu-dependent proteins and enzymes required for repair of radiation injury, including induction of metallothioneine (MT) synthesis, and recovery of radiation-induced immuno-incompetency. In this context, cytokines such as interleukm-1 OL-1), tumor necrosis factor, stem cell factor, and interleukin-12 have recently been suggested to restore function to tissues damaged by irradiation and protect mice from radiation lethality when given before irradiation [53]. In contrast, cytokines such as transforming growth factor I, interleukin-6, and interferon, given before irradiation, sensitize mice and increase radiation lethali.ty [54]. It is most likely that there are complex cytokine interactions relevant to the amount of cytokine nthesized and timing of release in responding to the degree of radiation injury.
Additional evidence for this comes from the correlation between survival of irradiated keratinocytes and the synthesis of IL-6 [55]. It is also known that changes in ceruloplasmin (Cp), a Cu-dependent multifunctional plasma enzyme, the plasma Zn containing a2-macroglobulin, and transferrin, the plasma Fe transporter, mediated responses to injury are regulated by IL-1 and other cytokines m facilitating tissue repair processes. Addition of inflammatory cytokines; IL-lt, IL-II or inorganic Cu to hepatocyte culture medium increased mRNA Ibr MT-I, MT-2, and Cp in a coordinated cytokine-activation of hepatic cells as an acute phase response to inflammation [56]. These observations offer a clearer understanding of the well-known cytokine-mediated acute phase response to inflammation and the increase in Cp as an acute phase response to irradiation-induced inflammation.
Following the observed radioprotectant and radiorecovery activities of Cu(II)(3,5-DIPS)4 as well as its facilitation of recovery of radiation-induced immuno-ineompetency, the question still remained as to whether or not there was any correlation between these observations and repair or recovery of radiationinduced histopathology. To address this need histopathologieal studies of spleen, bone marrow, thymus, and small intestine were conducted in parallel with immunological studies of animals exposed to LD50/0 irradiation (8.0 Gy, 1.55 Gy min"a) alone, treatment with 40 anol kg "1 Cu(II)2(3,5-DIPS)4 alone, or irradiated and treated with a single dose of vehicle or Cu(II)2(3,5-DIPS). Treatment with Cu(II)2(3,5-DIPS) brought about repair of radiation-induced histopathology in these tissues [see 48]. Signs of recovery were demonstrable by the 7th to 14th day after irradiation. These observations lead to the general conclusion that although Cu(II)(3,5-DIPS) does not prevent tissue injury when it is given before irradiation. Treatment with this chelate facilitates rapid tissue recovery from radiation-induced injuries. These recoveries from radiation-induced histopathology offer a biological marker for the re-establishment of the integrity of tissues known to be most vulnerable to irradiation.
The earlier review [see 48] also pointed out that Cu(II)2(3,5-DIPS)4 inhibits radiation-induced carcinogenesis and clastogenesis consistent with suppression of DNA replication and blockade of cell growth in the S phase as well as repair of DNA strand breaks. These effects are also consistent with reported anticlastogenic activity of Cu(II)(glycinate)2, observed as a rapid recovery of bone marrow cell radiation injury and associated repair of DNA [see 48]. They are also consistent with the prevention or repair of DNA strand breaks by Cu(II).(indomethaeinate)4 in phorbol-treated human leukoeytes and support Cu-dependent mechanisms for prevention or repair of radiation-induced DNA damage.
Whole body and local irradiation injury constitutes either systemic or local inflammation. Consistent with the antiinflammatory activities of Cu chelates [57], Cu(II)=(3,5-DIPS) has been found to be effective in facilitating repair of systemic inflammation caused by whole body irradiation (vide supra) and local inflammatory reactions. Ear swelling of female C3H mice treated 2 hr before porphyrin photosensitization and visible (480 to 645 nm) irradiation with 20, 40, or 80 btmol kg "1 Cu(II)2(3,5-DIPS)4 produced dose-related decreases in porphyrin-mediated photosensitized ear swelling of 25%, 50%, or 90%,reslxmtively, with a dramatic decrease in photosensitization-induced histopathology, suggested to be O-mediated inflammation due to activation of Xanthine Ox_idase (XO) [58]. This inflammation was inhibited by Allopurinol, an inhibitor of XO, and Verapamil, a Ca-channel blocker (vide infra) suggested to prevent the isoenzyme transformation of Xanthine Dehydrogenase (XD) to XO [59], the form of this enzyme that synthesizes H202 and 02. Consistent with this suggestion, ultraviolet epidermal cell injury of mouse ears (ultraviolet erythema, a recognized model of inflammation) was decreased with Cu:Zn2SOD treatment and inhibition of Cu2Zn2SOD by treatment with diethyldithiocarbamate exacerbated the "sunburn call" formation but did not change dtraviolet radiationinduced ear swelling [60]. Ultraviolet irradiated human endothelial cells elaborate nitrogen monoxide (NO) which may account for vasodilation and ear swelling as an inflammatory response [61] following activation of inducible Nitrogen Monoxide Synthase (NOS).
The observed decrease in skin Cu2Zn2SOD following a single ultraviolet irradiation was also reduced by pretreatment with liposomes containing Cu2Zn2SOD [62]. Increased ultraviolet radiation-induced keratinocyte proliferation was also found to be associated with a decrease in total SOD activity which was due mainly to a decrease in Cu2Zn2SOD [63]. Protection against ultraviolet radiation-induced chronic skin damage was also found with topically applied non-steroidal antiinflammatory agents [64]. These agents have been suggested to tbrm Cu chelates in vivo [57], which may account for their inhibition or repair of ultraviolet radiation induced injury.
Copper(II)2(3,5-DIPS)4 was originally examined for radioproteetant activity based upon reports that Cu2Zn2SOD had radioprotectant activity when given both before and after irradiation. However, subsequent reports suggested that superoxide dispmportionation by Cu2Zn2SOD did not account for its radioprotectant activity. Inactivated polyethylene-glycol derivatized Cu2Zng_SOD was as effective as the enzymatieally active derivative. The very real possibility that these proteins in extracellular spaces are in fact antigenic, although reduced antigenicity is always claimed, may lead to phagocyte elaborated IL-I and/or some other cytokine(s) that initiates immune and repair responses that accounted for the observed increase in survival [see 48].
The hypothesis that there are radiolytic losses of essential metalloelement-dependent enzyme cofactors [see 48] has merit and is consistent with the report that them was a 20% loss of both Cu2Zn2SOD and MnSOD in intestinal smooth muscle of rats immediately (1 hr) following irradiation (15 Gy). However, the dohn R.d. Sorenson et al.

Essential Metalloelement Chelates FaciBtate
Repair of Radiation Injwy activity of both enzymes increased over a 20 hr post-irradiation period to a level 45% higher than normal with a further increase in Mn SOD by 72 hrs post irradiation, a likely result of de novo synthesis in response to injury. It is noteworthy that Cu(II)(3,5-DIPS)4, a SOD-mimetic, was subsequently found to be more effective as a radioprotectant and radiomcovery agent than CuzZnzSOD and more effective than WR-2721 in reducing oxygen radical mediated colitis [65]. Adult CF male rots x-irradiated with 15 cGy see "1 day "1 for 7 days followed by 7 days rest for either 3 months or 6 months showed duration related radiation-induced patchy skin hyalinization, which became completely hyalinized during the succeeding 6 months without irradiation [66]. The concentration of Zn in skin of these rats remained normal in both irradiated groups but by the end of yr it had significantly (P < 0.001) decreased to 13 xg grn "a of tissue from 19 lag gm . Skin Fe was elevated in both irradiated groups and the elevation increased from 100 lag grn-of tissue with 3-mo irradiation to 140 lag grn l after 6-mo irradiation versus 68 lag grn "a for control skin. There was a further relative increase in skin Fe yr after irradiation to 129 lag grn " of tissue compared to 65 lag grn "1 for control skin (P < 0.001). It was concluded that chronic exposure to low dose irradiation possibly causes a redistribution of Zn and Fe with concomitant hyalinization of collagen. The observed skin collagen pathology and altered essential metalloelement contents were interpreted as results of chronic inflammation due to chronic irradiation [66]. These changes in skin essential metalloelement contents are consistent with essential metalloelement-dependent enzyme responses required to overcome radiation injury. Reduction in radiation-induced fibrosis with liposomal CuZn_SOD was studied using large white pigs ,/-irradiated with a single dose of 160 Gy delivered to the skin. Treatment with 0.3 mol Cu2ZnzSOD applied 2 times per week for 3 wk caused shrinking of irradiation-induced fibrosis in all treated pigs. Scar regression was found to be 45% in length and width, 30,4 in depth, and 70% in area and volume. Histologic examination showed normal muscle and subcutaneous tissue surrounding the residual scar. This replacement of scar tissue by normal tissues and a 50O/ decrease in the linear dimensions of the scar were comparable to results obtained in a previous clinical trial of liposomal CuZn_SOD treatment [67]. Over expression of Cuv_ZnSOD or MnSOD increased survival of 0.2 to 1.2 Gy (0.3 Gy min-) xirradiated CHO cells and cells over-expressing MnSOD had greater survivals that those over-expressing Cu2Zn,,SOD [68]. As pointed out earlier [see 48] the SOD-mimetic reactivity of Cu(II)2(indomethacinate)4 was credited with the prevention of DNA strand-breaks in human leukocytes stimulated with phorbol-diester.
This prevention or facilitated repair of DNA was observed over a concentration range of 2 to 9 btM Cu(II)2(indomethacinate)4.
Another possible mechanism offered to account for the radiopmtectant and radiorecovery activities of Cu chelates involves Cu-dependent nuclear metabolism. It is known that 20% of cellular Cu is found in the nucleus. Copper has the highest bonding affinity for nucleic acids of all essential metalloelements. Nucleic acid phosphate and base donor sites yield Cu chelates, which are much more stable than Cu chelates of most amino acids and near the stability of protein chelates. This stability has been used to support suggestions of intrastrand and interstrand DNA bridging, which was recognized early as having a role in stabilization of the Watson-Crick double helix [see 48]. Higher physiologic concentrations account for inhibition of ribonuclease syntheses: initiation, nucleotide condensations, and release of newly formed mRNA. However, the original observation that injection of Cu(II)CI2 into rats induced the synthesis of mRNA coding for the synthesis of MT has been explained as due to the formation of a Cu-dependent DNA bonding protein ACE1 or CUP2, the protein products of S. cerevisiae (ACE1) and E. coil (CUP2) genes, which recognize upstream activation sequences required for active transcription of CUP1, a MT gene as pointed out earlier [see 48] ,and subsequently [69]. Chaperones such as hAtoxl are Cu bonding proteins suggested to have roles in mediating Cu homeostasis in many cell types as cytosolic Cu-transporters, enabling the utilization of Cu in tie novo synthesis of Cu-dependent enzymes and proteins [70]. Since radiation injury and injection of Cu chelates induce MT synthesis and increased MT synthesis correlates with increased survival, Cu chaperones such as Atoxl, which mediate the role of Cu in transcription of Cu2Zn2SOD and the anti-apoptotic bcl-2 genes [71], may represent important nuclear chemical responses in overcoming radiation injuB..'.
Nuclease-Mimetic reactivi.ty of Cu chelates has been recognized as valuable footprinting reagents useful in revealing details of RNA polymerase bonding to a lac promoter [see 48]. Predictable chromatin DNA cleavage by a chelate due to preferential bonding of the chelate to DNA spacer segments is also a useful characterizing feature. Interpretations that these and other DNA strand scissions resulting from Cu chelates ae damaging [72,73] without recognizing that these scissions may be due to expected Cu chelate [74] or other essential metalloelement chelate [75] catalyzed diphosphate ester hydrolyses, which may be viewed as endonuclease-(or exonuclease-) mimetic reactivity, merit reconsideration. More recently, the antineoplastic action of the copper complex of Tambjamine was suggested to be due to DNA nuclease reactivity [76]. Like many Cu chelates, Cu(II)(3,5-DIPS)4 has SOD-mimetic reactivity. While formation of ternary protein-Cu(II)z(3,5-DIPS)4 chelates formed with collagen or albumin have slightly less SOD-mimetic reactivity due to protein bonding to a free Cu bonding site [77], it is the formation of, as yet not unequivocally demonstrated, ternary albumin chelates formed in vivo [78,79] that were suggested to account for the observed rapid and pervasive tissue distribution of 67Cu and 1C labeled 3,5-DIPS within 0.5 hr following administration of 67Cu(IIh[3,5-DIP(Carboxy-1C)S] [80].
Dietary supplementation with arginine, the substrate for NOS, required for the synthesis of NO has been found to accelerate intestinal mucosal regeneration and enhance bacterial clearance following radiationinduced rat enteritis [81]. Rats fed diet containing 4% arginine for 7 days following non-lethal local abdominal x-irradiation (11 Gy, 0.14 Gy min") had significantly (P<O.001) increased mesenteric lymph node mass, decreased number of lymph node Gram-negative enteric bacilli and facultative anaerobic bacteria, CFU gm" of lymph node, increased mucosal thickness, and jejunal and ileal villi height and number, as well as number of mucous cell villi compared to rots fed a diet containing 2% arginine or 4% glycine.
Copper(II)(3,5-DIPS) has been suggested to facilitate NO mediated responses to inflammato diseases [82,83]. However, it has also been found to decrease synthesis of NO by down-regulating nitric oxide synthase (NOS) enzyme activity which may account for the antiinflammatory activities of this and other Cu chelates as well as repair of radiation injury [82,83]. This is an eially interesting possibility since Cp, which increases in plasma following irradiation, has recently been reported to decrease NOS activity when incubated with endothelial cells. There was a time-dependent increase in cytosolic and particulate bonded copper, and this effect could be reproduced by incubation with Cu(II)(histidinate),,_ but not with Cu(II)CI2. which primarily bonded to all particulate fractions and less so to cytosolic components [84]. In addition, CuZn2SOD has recently been shown to release NO from S-nitrosothiols in the presence of glutathione [85] suggesting that Cu2Zn.SOD might have a role in mediating constitutive or inducible NOS and facilitating NOmediated physiological functions as well as mediating rat mucosal regeneration and bacterial clearance in radiation enteritis [81 ].
Recently a domain of Cp, distinct from the ferro-oxidase domain, has been reported to act as a glutathione-dependent pemxidase [86] and a glutathione-dependent alk3'l hydroperoxidase [87], which offers an interesting accounting for a plasma Cu-dependent agent responsible for removal of lipid hydroperoxides. Cemloplasmin has also been recently identified as a plasma Pituitary Adenylate Cyclase-Activating Polypeptide (PAC-AP) 1-38-bonding factor [88]. This factor is an alpha amidated peptide, one of many amide terminus neuronal hormone peptides synthesized by Cu-dependent x-arnidating mixed function oxidases [89]. PAC-AP has more recently been reported to prevent cytokme-induced pancreatic B-cell cytotoxicit3" by inhibiting inducible NOS expression in BTC cells [90]. The PACAP activity of Cp has also been reported to decrease cytokine-induced cytotoxicity by inhibiting inducible NOS in pancreatic I cells [91]. These observations offer an accounting for the radioprotectant and radiorecovery activities of Cu(II),,(3,5-DIPS)4. since Cu(II)2(3,5-DIPS)4 has been reported to down-regulate, but not inhibit enzyme activib', NOS and decrease the synthesis of NO [81,82]. Alternatively, this Cu chelate as well as others may form ternar3 nitrogen monoxide chelates (ON-CuL2) that have a role facilitat.ng NO dependent processes [81,82]. The possibility remains that reported therapeutic actions of Cu chelates may be due to only a small portion of the admimstered dose bonded to serum albumin and transported to the site of action. That is, an effective antiinflammatory dose of Cu(II)2(3,5-DIPS)a is tmol per kilogram of body mass. This represents 1019 molecules per 70 kg adult treated with a supposed dose of 70 txrnol. If only 0.01% of this dose was distributed throughout the body due to losses including excretion, each of the estimated 101" cells in the adult body could experience contact with more than 1,000 molecules of this chelate. Since there is always ,an inflammatory-response mediated vasodilation at the site of disease-affected cells and greater perfusion of these affected cells by plasma filtrate, the concentration of drug in the environment of disease-affected cells could actually be greater than 1,0 molecules per cell. Smaller daily doses ranging down to 0.7 tamol per adult, which could be a more likely dose, would provide 10 molecules per cell with the loss of 99.99% of the administered dose. Such losses may be typical for all administered drugs.
Radiation Protection trod Recovery with Iron Chelates.
Radiorecovery of mice treated with this chelate and irradiated with smaller doses of irradiation remain to be determined.
Gamma irradiated (7 Gy, 2.9 Gy min1) young adult male DBA/2 mice treated with 16 mol kglip Fe(III) hematoporphyrin chloride (FeHp) either and 4 days or 1, 4, and 7 days niter irradiation had significantly (P < 0.025) increased spleen colony forming units (CFU-GM) by day 14 following irradiation [92]. Mice treated on days and 4 after irradiation had significantly (P < 0.025) increased numbers of spleen cells by day 17 post-irradiation. IronHp treatment on days 1, 4, and 7 also caused a significant (P < 0.005) reduction in bone marrow cell number by day 14 and other post-irradiation periods of measurement. There was no increase in bone marrow CFU-GM observed for any treatment. Mice treated 24 hr before irradiation and 48 hr or 48 and 96 hr niter irradiation produced significant (P < 0.001) increases in total spleen cell number and spleen CFU-GM by day 15 post-irradiation. The single treatment t'ter irradiation caused a 5-fold larger increase in spleen CFU-GM number than non-irradiated or irradiated control values measured on day 15. The only significant (P < 0.01) change in bone marrow cellularity was a decrease in cell number for mice treated 4 hrs before and 48 and 96 hrs d'ter irradiation. There were no significant changes in bone marrow CFU-GM number at any time of measurement on 11, 14, and 17 days post-irradiation, relative to irradiated control mice. These data were interpreted as due to hematostimulatory activity of FeHp with stimulation occumng following radiation-induced immunosuppression. Enhanced hematopoietic recovery was observed for spleen but not bone marrow of irradiated mice treated with FeHp. Treatment may have also increased the mobilization of progenitor cells from bone marrow to the spleen in addition to the FeHp stimulatory effects on spleen CFU-GM. Interestingly, the FeHp-mediated spleen hematopoietic response was greatest in mice treated 24 hrs before irradiation. There was no "protection" but resting hematopoietic stem cells may have been reduced to begin the cell growth cycle and division, while actively dividing cells would have been at gater risk to radiation injury and cell death.

Radiation Protection and Recovery with Manganese Chelates.
The earlier review [see 48] cited a number of reports that inorganic Mn(II)CI2 given sc in the radioprotectant paradigm before irradiation increased survival and enhanced survival when co-administered with aminothiol radioprotectants. Following these reports Mn(II)C12 was shown to repair radiation-induced skin and intestinal inflammation in mice [93]. Treatment with 180 lamol Mn(II)CI2 kg given ip to male C3H mice 24 hr before a single 20 to 52 Gy (5 Gy min"1) local paw x-irradiation or 12 to 21 Gy abdominal irradiation fed to repair of acute intestinal and skin radiation-injury. Skin and intestinal MT was not increased by administration of MnCI2 although a six-fold increase was found in liver. These authors failed to point out that the increase in liver MT was associated with the increase in survival of mice treated with Mn(II)C12.
Mechanistic Modes of Mn Chelate Action.
A possible Mn-(3,5-DIPS)2 mechanism of action in overcoming inflammation due to radiation injury is the dovn-regulation of NOS when excess NO is produced as part of the inflammatory response [82]. Alternatively, activation of Mn-dependent guanylyl cyclase [95], the receptor for NO, by Mn-(3,5-DIPS)z may serve to restore normal physiologic functions involving NO-mediated processes. This understanding is consistent with the suggestion that a ternary NO-Mn-(3,5-DIPS)z complex might form in vivo [82] and have roles in mediating NO function [83].
The Mn-Desfemoxamine (MnDFO) SOD-mimetic chelate has also been found to be effective in protecting against riboflavin-mediated ultraviolet (365 nm, 2 to 9 J cm"2) phototoxicity [96]. Photohemolysis of erythrocytes was decreased with 500 vl MnDFO. Treatment with 50 or 100 ONI MnDFO did not change 3H-Thymidine incorporation in irradiated (5 J cm"z) GH-U1 human urinary bladder carcinoma cells or irradiated (10 J cm"2) phytohemaglutin-stimulated lymphocytes compared to radiation alone. Higher concentrations of MnDFO were toxic to these cells.
Hahn et al. [97] and Dart et al. [98,99] suggested that MnDFO is superior to Cu chelates as an SODmimetic because Cu chelates dissociate in the presence of protein. This suggestion was offered even though Cu chelates are much more effective in disproportionating O2" at an appropriate physiological pH and more effectively treat many disease states (see 2). They overlook the fact that Mn chelates also dissociate in the presence of protein or other biochemically important macromolecules via ligand exchange. However, these losses may not prevent their use in treating diseases (vide supra et infra). More importantly they overlook the possibility that the presence of protein such as albumin can lead to temary Cu or Mn chelate formation with enhanced stability and serve as Cu or Mn chelate transporting agents in vivo [77][78][79]. Consequently, the view that Cu or Mn chelates can not be effective SOD-mimetics in preventing Oz-mediated pathology, despite many observations to the contrary is seriously flawed. Finally, Mn chelates will not serve to facilitate the activation of Cu-dependent enzymes nor will Cu chelates facilitate activation of Mn-dependent enzymes.
The SOD-mimetic reactivity of Mn(III)tetrakis-l-methyl-4-pyridylporphyrin pentaehloride [Mn(III)TMPyP] was recently suggested to aount for the restoration of NO-mediated neurotransmission and serves as a lead compound in the development of SOD-mimetics as therapeutic agents for the treatment of neuropathies associated with oxidant stress [100].
While increasing the dose of Cu(II)2(3,5-DIPS)4 from 10 to 40 pmol kg " increased survival, increasing the dose of Mn-(3,5-DIPS)2 fi'om l0 to 40 pmol kg "l did not increase survival and increasing doses of Mn-(3,5-DIPS)2 beyond 20 lamol kg 1 given in combination with Cu(II)z(3,5-DIPS)a were associated with decreases in survival or no increase in survival due to combination-treatment. This apparent negative effect on survival associated with increasing doses of Mn-(3,5-DIPS)_ given in combination with Cu(II)9(3,5-DIPS)4 may be due to an interference of Mn in de novo synthesis of Cu or some other metalloelement-dependent enzymes wherein the incorporation of Mn leads to a reduction in activity of non-Mn metalloelement-dependent enzymes requiring a metalloelement other than Mn for activation.
Irradiation-Induced esophagitis, micro-ulceration, and esophageal stricture as well as cytokine mRNA elevations and squamous cell apoptosis were dramatically decreased in male C3H/HeNsd mice with intraesophageal instillation of mouse MnSOD-plasmid/liposome (SOD2-PL) 24 hr before 35 or 37 Gy local esophageal x-irradiation (58). Mice bearing orthostatic thoracic tumors treated with mouse SOD2-PL also evidenced esophageal transgenic SOD2 mRNA [102]. These results were suggested to provide support for human SOD2-PL gene therapy in a fractional irradiation paradigm to avoid acute and chronic radiationinduced side effects in a human clinical trial of pulmonary ttunor irradiation 102]. Increased stuwival was also observed at both levels of irradiation. A significant increase in surviving fraction (1.0, P 0.0291) versus 0.5 for control liposome treated mice was observed for 35 Gy irradiated mice treated with 100 tg SOD2-PL/mouse given by the mtra-esophageal route of administration with a significant (P 0.015) decrease in esophagitis in this 30-day study. Mice irradiated with 37 Gy and treated with 200 xg SOD2-PL/mouse or 10 xg SOD2-PL/mouse by intra-esophageal instillation experienced increased surviving fractions of 0.5 (P 0.05) or 0.7 (P 0.0119) respectively compared to non-treated irradiated mice in this 60-day study 102]. While mtra-trachael instillation of transgene before irradiation protected C57BL/6J mice from whole-lung irradiation-induced alveolitis/fibrosis, there was no significant lung protection in SOD2-PL treated SP1-SOD2 or FeVI3/NHsd transgenic mice. Over expression of either human SOD2 or murme Sod2 in the lungs of these transgenic mice did not provide local 11 to 15 Gy irradiated whole-lung or alveolar type II cell protection [103]. Over expression of SOD2 in SP-1 SOD2 mice may have made these mice more sensitive to radiation injury. However, these studies may have been seriously flawed since mice developing pulmonary distress were killed, preventing the possible observation that treated mice might have overcome this radiation-induced inflammatory complication.
Radiation Protection and Recovery with Zinc Chelates.
As pointed out in our earlier review [see 48] treatment with Zn compounds dramatically increased survival of lethally irradiated mice. In the interim it was shown that fractional whole-body /-irradiation of male BALB/C mice given a total doses of 0.5, 1, or 1.5 Gy induced an increase in DNA-protein cross-links in the thymus, spleen, brain, and liver. However, when these mice were given drinking water containing 154 Zn(II)SO4 for 30 days DNA-protein cross-links were dramatically decreased to levels observed in all of these tissues from non-irradiated mice 104].
Fibroblasts cultured in medium containing 100 p_M Zn(II)CI2 had repaired ultraviolet radiationinduced DNA strand-breaks and delayed in apoptotic loss of nucleosomes [105] Cells cultured with 100 tM Zn(II)C12 and irradiated with 1.4, 2.9, 3.6, or 5 J/cm 2 actually evidenced more double-stranded DNA than nonirradiated cells. This result suggests that Zn(II)CI2 prevents and/or repairs DNA strand-breaks Similarly, apoptosis was always less in Zn(II)CI2 supplemented cells at all times of measurement; 0, 3, 6, 8, 11, 12, and 18 hrs after irradiation consistent with prevention and/or repair of irradiation-induced injury leading to apoptosis. Gamma irradiation-induced thymocyte DNA strand-breaks and apoptosis were synergistically decreased with combinations of Zn(II)SO4 and diethyldithiocarbamate, a compound known to form Zn(II)(diethyldithiocarbamate) 2 106].
Since addition of Zn (0.15 pM) to culture medium did not protect human' melanoma tells tlu'ough a radiation dose range of 1.5 to 8.0 Gy (0.85 Gy minl). It had been suggested earlier [see 48] that the protective effect of Zn was due to the facilitation of some homeostatic mechanism which was suggested to be induction of MT synthesis [see 48]. Studies of MT gene regulation identified the involvement of bacterial lipopolysaccharide, IL-I, interferon, and other immunostimulants with control of MT syntheis in the liver. These observations coupled with the observation that IL-1 has radioprotectant activity [54] support the hypothesis that MT synthesis has a role in reducing radiosensitivity. Induction of MT synthesis is a physiological response to oxidant stress as described for IL-1 or endotoxin-mediated responses to this stress as well as other cytokine-mediated responses to radiation injury. [58,59].
As pointed out earlier [see 48] Zn(II)(aspartate), is known to have radioprotectant activi:. An important question as to whether or not this compound protected neoplastic cells from radiation inju.w was answered when ip treatment with 90 pmol kg "1 Zn(lI)(aspartate)2 did not inhibit 7-ray radiotherapcutic reduction of tumor volume using three adenocarcinomas of the colon or Ewings sarcoma implanted immunosuppressed mice [107]. Zinc(II)(aspartate). also significantly reduced radiation-induced decrease in hematocrit and thrombocyte, erythrocyte, and leukocyte numbers, an indication of spleen and/or bone man'ow precursor cell sparing effect or rapid recovery of immunocompetence. Zn(II)(aspartate)_ permitted a greater reduction in tumor volume than cysteamine [mercaptoethylamine (MEA)], mET, and WR-2721 and it was as effective as a syngeneie bone marrow transplant [107].
Mechanistic Modes of Zn Chelate Action.
Since Zn chelates have no SOD-mimetic reactivity, activation or synthesis of Cu2Zn2SOD, MT, or other Zn-dependent enzymes including endonucleases and other known and suspected Zn-dependent transcription and translation processes [110] must at least partially account for their radioprotectant or radiorecovery activity. Another partial accounting may be attributed to protection of spleen and bone marrow stem cells by decreasing DNA replication and blockade in the S growth phase as observed tbr CHO cells treated with Zn(II)CI. as pointed out earlier [see 48]. A 100 pmol kg " dose of Zn(II)-Desferrioxarnine [Zn-DFO] produced 60% survival in whole body LD50/30 /-irradiated female C3H mice treated 0.5 hr before irradiation while Desferioxamine had no radioproteetant activity [111]. There was no radioprotective effect observed in studies of cultured V79 Chinese hamster cells. However, Zn-DFO concentration dependently prevented radiation-induced plasmid DNA strand-breaks through a concentration range of 0.5 to 2 mM.
Feeding an "antioxidant" diet containing 55 trnol Zn(II)(gluconate)2 kgday l to C57BL male mice for 45 days before irradiation(6 Gy, 1.8 Gy min") caused an inhibition of TRPM-2, a marker gene for apoptosis expression both before and after irradiation [112]. The bcl-2 mRNA, which is involved in the prevention of apoptosis, and splenocyte number increased steadily after irradiation. The expression of sod-I gene increased and remained elevated for 24 hr, the last time of measurement after irradiation, and synthesis of the eat (catalase) gene increased slightly shortly after irradiation and decreased by 3 hr after irradiation.
Preliminary studies under the same experimental conditions revealed a decrease in frequency of radiationinduced mutations as evidenced by changes in the hypoxanthine-quanine phosphoribosyl-transferase locus of splenocytes from mice fed the antioxidant diet containing Zn(II)(gluconate)2. The maximum antimutagenic effect was detected by the 6 th week of diet administration.

Radioprotectant and Radiorecovery Activity of Calcium-Channel Blockers.
New evidence provided by Floersheim and his colleagues document radioprotectant and radiorecovery activities for some Ca-channel blockers [113][114][115][116]. These reports are consistent with the earlier suggestion of Athar et al. [59] that Veraparnil, a Ca-channel blocker, prevents radiation injury-induced conversion of Xanthine Dehydrogenase to Xanthine Oxidase, which synthesizes 02-.
Isradipine, another dihydropyridine Ca-channel blocker, given at a dose of 74 tmol kg " ip before irradiation (9 Gy) produced 58% survival. Treatment with 278 ktmol kg "1 Nitrendipine (a dihydropyridine Cachannel blocker), ip, produced 29% survival. Verapamil, a substituted aminopropylnitrile Ca-channel blocker, given at a dose of 35 pmol kg "1 ip and Flunarizine, a piperazine Ca-channel blocker, given before irradiation (9 Gy) at a dose of 62 nol kg "1 orally did not produce any increase in survival compared to the vehicle-treated group [113]. Based upon the efficacy of Verapamil in facilitating repair of photosensitization-radiation injury and its prevention of the XD to XO conversion as reported by Athar et al. [58,59], this mechanistic feature of Ca-channel blocker activity must be taken into account in examining their potential for radioprotectant activity. It would also be useful to examine this mechanistic aspect of all radiorecovery agents.
Floersheim suggested that Ca-channel blockers might also act by blockade of cell death due to Ca influx into cells following radiation injury [113]. However the antioxidative effects of Ca-eharmel blockers via scavenging of free radicals was suggested as an alternative mechanism of action, which could involve stable essential metalloelement chelate formation following hydrolysis of ester functional groups in vivo. Radical scavenging reactivity of ammothiol radioprotectants and formation of their disulfides was also suggested as a mechanism of their action in overcoming radical-mediated radiation injury [113]. A chelate mechanism for radical scavenging by aminothiols is also plausible. Variations in activity of Ca-eharmel blockers were suggested to be due to variations in bioavailability and receptor-bonding interaction [113].
Benzothiadiazepines and dihydropyridine Ca-channel blockers may also serve as chelating agents and thus facilitate delivery of essential metalloelements to cells for de novo synthesis of essential metalloelementdependent repair enzymes. Calcium-Channel blockers have also been reported to decrease radiation-induced esophagitis as a consequence of thoracic irradiation. Variation in blood flow was proposed to explain the modified x-ray sensitivity of mouse tumor [113]. Calcium-Channel blocker-induced hypotension and tissue anoxia was negated as the cause of radioprotectant activity of these agents, since other agents causing these conditions had no radioprotecting activity [113]. Subsequently Floersheim reported [114] that 9 mol Nifedipine kg t given ip 3 hr before LDl00 irradiation (9.8 Gy, 0.9 Gy min") produced 83% survival. Again, even though the radiation dose was a LD0oco dose, the 96% ethanol vehicle produced 61% survival while an 18% ethanol vehicle produced 28% survival.
Vempamil, given to these mice at doses of 35 lamol kg"tip or Flunarizine, a piperazine Ca-channel blocker, given to these mice at doses of 62 trnol kg "t ip at 30 or 120 min tiller irradiation did not produce any increase in survival compared to the 00/ survival for vehicle-treated mice 114].
Combinations of Diltiazem and Nifedipine were also studied using the factorial paradigm. A synergistic increase in survival was found for increasing active doses of Nifedipine and Diltiazem from 61 to 122 prnol kg when given before irradiation [114]. No [114]. Koch [115] suggested that enhanced local blood flow caused by Ca-channel blockers might be responsible for increased radiosensitivity due to the oxygen effect. The exchange by Koch [115] and Floersheim [116] offered a number of spectflations concerning Ca-channel blockade in radiosensitization and radioproteetion. These related to dose, chemical class, a possible role for small molecular mass solvent molecules, as well as effects on metabolizing enzymes.
Interestingly, calcium was found to be elevated in heart, liver, lung, spleen, and kidney of adult male albino rats but decreased in these tissues of female rats two months after 5 Oy ,t-irradiation [117]. While cardiac Fe was elevated, Fe content decreased in all other tissues of male rats. There were no changes in heart and lung Fe in female rats while liver, spleen, and kidney content decreased in female rats. Zinc was elevated in the heart, decreased in lung and spleen, and unchanged in liver and kidney of both male and female rats [117].
Radioprotectant Activity of Serotonin and Acyl Melatonin Homologs.
A review of Melatonin, N-acetyl-5-methoxytryptamine, antioxidant effects pointed out that Melatonin is relatively non-toxic and protects against ionizing radiation injury [118]. This review cited the report of Blickenstaff et al. [119] as having shown that Melatonin, the acetylated methoxyether of Serotonin, 5hydroxytryptarnine, produced 75 % survival in LD00/30 9.5 Gy whole-body irradiated male Swiss ND4 mice. This was an increase in radiation protection compared to Serotonin, which produced only 20 % survival in the same study. Intraperitoneal treatment of these mice with soybean oil solutions or suspensions of 1.1 mmol kg 1 acetyl (Melatonin), propionyl, butanoyl, pentanoyl, hexanoyl, oetanoyl, decanoyl, or hexadecanoyl analogs of 5-methyltryptamine 30 rain before irradiation produced survivals of 0 %, 43 % (P 0.0045), 32 %, 75 %, 95 %, 65 %, or 48 % respectively. In this experimental paradigm a 0.78 mmol WR-2721 kg 1 treatment produced 50 % survival. Radioprotectant activities of all Melatonin analogs were significantly different from vehicletreated mice and the activity of Melatonin was about equal to that of WR-2721. The increase in radioprotectant activity was greatest for the hexanoyl and octanoyl derivatives, showing an increase in activity with increasing lipophilicity. However, further increasing lipophilieity with the formation of still longer chain amides lead to a decrease in activity, as is usually the case when lipophilicity is increased beyond some optimal value. The observed radioprotectant potency as well as a lack of Melatonin toxicity was suggested to support further study of Melatonin and these acyl homologs in circumstances of human radiation injury [118].
These compounds can participate in coordinate-covalent bond formation with essential metalloelements. The considerable enolic character of the amide carbonyl group and the proximity of the weakly acidic indole amine offer possible coordination sites.
Vijayalaxmi et al. found that treatment hr before LD5030 8.15 Gy whole-body irradiation with ip doses of 0.55 or 1.1 mmol Melatonin kg " suspended in soybean oil produced survivals of 50 % or 85 % (P 0.0080) respectively in CD2 mice versus 50 % survival for vehicle-treated mice [120].
Karbownic et al. [121] recently reported that 440 tmaol Melatonin kg " given as a soybean oil suspension ip to male Sprague-Dawley rats four times at 120, 90, 60, and 30 rain before whole-body 8 Gy irradiation completely prevented the formation of 8-hydroxy-2'-deoxyguanosme and mierosomal membrane rigidity in hepatocytes. Both of these damages are consequences of ionizing irradiation due to DNA or protein Repair of Radiation Injury and lipid damage. It is noteworthy that these repairs of radiation injury were observed as soon as 12 hr after treatment and irradiation.
Using the same experimental paradigm that allowed the observation of radioprotectant activi.ty for Melatonin homologs, Blickenstaff et al. [122] found that a series of substituted anilines had radioprotectant activity. Intraperitoneal treatment of male Swiss ND4 mice with soybean oil solutions or suspensions of 780 xmol kg 1 [122].
Curcumin, a I-diketone {1,7-bis(4'-hydroxy-3'-methoxyphenyl)-l,6-heptadiene-3,5-dione}, is a yellow major component of Turmeric, a spice prepared from dried rhizomes of Curcuma longa L. Tumeric is widely used to season foods in Asian countries and in particular, Indian Ctna3,. It is of particular interest to note that Curcuma longa L is a fungus and fungi are well known for their high copper content. Since 13diketones are well known chelating agents [123,124] and this fully conjugated [5-diketone is likely to form particularly stable Cu chelates as a result of the extended conjugation m this ligand, it may be that this ligand exists as a Cu(II) chelate in fungi.
In 1993 Abraham, Sarma and Kesavan reported that Curcumin decreased chromosomal damage in ,irradiated mice [125]. A single oral administration of 27.2 nol Curcumin kg to male Swiss albino mice 2 hr after whole-body 1.15 Gy irradiation significantly (P 0.01) reduced frequencies of micronucleated polyehromatie erythrocytes (MPCEs) in bone marrow samples taken 24 hr after irradiation. Oral administration of 13.6, 27.2, or 54.4 xmol Curcumin kg t 2 hr before irradiation produced a dose related decrease in MPCEs in bone marrow samples taken 24 hr after irradiation. Oral treatment with 27.2 tmol Curcumin kg 1 2 hr before irradiation produced significant (P 0.05 to 0.01) time-dependent decreases in MPCEs in bone marrow samples taken 24, 30, and 48 hr after irradiation. These results suggest that Curcumin is useful in facilitating recovery from radiation injury.. Subsequently, lnano et al. [126] reported that feeding Curcumin decreased the incidence of marnmm3' tumor m rats. A 1% Curcumin diet fed to 2.6 Gy whole-body irradiated pregnant Wistar-MS rats just prior to pup birth (a mammary tumor imtiating procedure) and then implanted with diethylstilbestrol sustained release pellets for 12 mo following weaning (a mammary tumor promoter regimen) caused a dramatically decreased incidence of mammary tumor. Tumor formation decreased from 84.6 % for the control diet led group versus a 28 % incidence for the Curcumin fed rats. Feeding the diet containing Curcumin significantly (P<0.001) reduced the number of rats with tumors and the number of tumors per rat and increased the latency period for mammary tumor formation (P< 01001). Using this same pregnant Wistar-MS rat model of marnmm" tumorogenesis and feeding a 1% Curcumin diet for a brief period from day-11 of pregnancy to day-23, the day of birth, with 1.5 Gy whole-body irradiation on day-20, prior to birth of the rat pups, again significantly (P<0.0001) decreased the number of dams with mammary tumors from 19 (70.3 % incidence) for the control group to 5 (18.5 % incidence) for the Cureumin fed rats. This Curcumin diet protocol also significantly (P<0.0001) delayed development of mammary tumors in Curcumin fed rats. Eventually mammary tumors did develop during the last two months of this 12-mo study [127]. Curcumin was fed for only 10 days in this study while it was fed for yr in the first study (82). Thus further documenting and extending the application of dietary Curcumin in preventing irradiation-induced mammary tumors.

CONCLUSIONS
Understanding essential metalloelement metabolism and its role in responding to radiation injur." as mediated by immunomodulating cytokines does offer a renewed and potentially more effective approach to recovery from all radiation syndromes. Copper, Fe, Mn, and Zn chelates have radioprotectant activity based upon results obtained with treatment before radiation injury and some have been observed to have radiorecovery activity based upon results obtained with treatment M'ter radiation injury. These compounds do not prevent radiation injury however they do facilitate repair and recovery from radiation injury. They are also far less toxic than existing radioprotectants. Existing radioprotectants have not been to shown to have radioreeovery activity. New chelates with increased efficacy and still less toxicity may be found using imaginative synthetic approaches to new ligands based upon existing compounds such as amino acids, salicylates, as well as radioprotectant aminothiols Ca-channel blockers, acyl Melatonin analogs, substituted anilines, and Cureumin. These chelates will also have potential for improving therapies of inflammatory diseases, ulcers, seizures, neoplastic diseases, diabetes, and pathology associated with ischemia-reperfusion Combination treatment with essential metalloelement chelates and aminothiols, Ca-channel blockers, acyl Melatonin homologs, substituted anilines, and I-diketones such as Curcumin also offer an approach to improving radiation recovery and treatment of other recognized disease states. Combinations of some of these agents with essenti'al metalloelement chelates may lead to the development of new physiological, biochemical, and pharmacological approaches to overcoming many disease states.