Hemoperfusion for ARS treatment .
Leukopenia is an essential part of the clinical course of acute radiation sickness and is a side effect of anti-cancer treatment. In both situations, the main factors which determine the survival are the degree of bone marrow suppression and gastrointestinal tract damage due to the presence of a large pool of fast-dividing cells. Leuko- and neutropenia are main limiting factors which may contribute to chemotherapy failure. Hematopoietic cytokines the part of conventional therapy in this field, but their effects require boosting. That is why the use of means and methods of adsorption therapy is considered promising. Sorption therapy creates a basis for sorption detoxification, a doctrine of curative measures directed to the removal of toxic endogenous or exogenous compounds from body fluids. The most widely used types are the purification of blood or its components (hemosorption), oral administration of sorption materials (enterosorption) and application-sorption therapy of wounds and burns. In this chapter, the results of early and recent research and prospects for the use of carbon adsorption therapy for the treatment of acute radiation sickness and cytostatic myelosuppression are discussed.
- ionizing irradiation
- anti-cancer chemotherapy
- granulocyte colony stimulating factor
- application-sorption therapy
The danger of acute and chronic radiation injuries, which provoke leukopenia, is not just a myth today. The explosion at Unit 4 of Chernobyl Nuclear Power Plant (NPP) in 1986 showed how unprepared people were to such a problem. The collective dose of irradiation for liquidators (clean-up workers) was huge; no one knows the exact numbers (all dosimetric equipment measured only gamma irradiation). And until today, about five million people, who live in areas of Belarus, the Russian Federation and Ukraine, which are contaminated with radionuclides, still experience the consequences of pollution [1, 2, 3]. An earthquake and tsunami struck Fukushima Dai-ichi NPP in 2011 contaminated the soil and water with radioactive cesium, iodine, etc. It poses significant risks of exposure to the residents [4, 5]. Terroristic threats or military conflicts with the use of radioactive weapons could be considered as a potential risk of injuries also.
One more source of contact with myelosuppressive factors is radiation therapy, which is routinely used in oncology (up to 70% of patients with malignant tumors are treated with) as well as anti-cancer chemotherapy with cytostatics [6, 7, 8]. Medical use of radiation accounts for 98% of the population dose contribution from all artificial sources and represents approximately 20% of the total exposure. Annually worldwide, more than 3600 million diagnostic radiology examinations are performed, 37 million nuclear medicine procedures are carried out and 7.5 million radiotherapy treatments are given . In spite of side effects, the concomitant use of radiotherapy and chemotherapy resulted in significantly improved clinical outcomes [10, 11, 12]. Different radiomimetics have effects similar to ionizing irradiation. Among them, a lot of anti-cancer drugs and leukopenia is a common side effect of dose-dense and dose-intense tumoricidal chemotherapy.
The organs and tissues with high speed cell proliferation is the most sensitive for radiation- and radiomimetic damage. Leukopenia, because of aggressive direct ionizing irradiation or anti-cancer chemotherapy with cytostatics, is an important prognostic factor for overall survival [13, 14]. The association between chemotherapy-induced leukopenia and clinical outcome has been reported for several types of cancer. The development of such health impairments gains more and more attention, especially after the success of modern techniques such as stem cell transplantation and cytokine treatment to restore hematopoietic functions. But even now, it is not enough for the treatment of acute radiation sickness.
In last decades, we observe combined injury by ionizing radiation and toxic effects of xenobiotic, thermal burns, mechanical trauma, etc. Despite significant achievements in oncology, precise and targeted irradiation of tumors, the development of effective means for enhancement of bone marrow cell and peripheral blood cells proliferation (granulocyte colony stimulating factors (G-CSF), erythropoietin, interleukin-11 and others), the problems of fighting the negative consequences of ionizing radiation and radiomimetics remain very important.
In this chapter, the results of early and recent research and prospects for the use of carbon adsorption therapy for the treatment of myelosuppression caused by acute radiation sickness and cytostatics use are discussed.
2. About radiation injuries
Acute radiation syndrome is a definition to reflect severe damage to specific organs that occurs because of whole-body or significant partial-body irradiation greater than 1 Gy, over a short time period (high dose rate) . The main syndromes are hematopoietic (doses >2–3 Gy), gastrointestinal (doses 5–12 Gy) and cerebrovascular one (doses 10–20 Gy) . Depending on exposed and absorbed doses and its duration, cells exposed to ionizing radiation or radiomimetics present DNA mutations, apoptosis, necrosis, chromosomal aberrations or increased mutation frequency [17, 18]. The most profound injury is to lymphoid organs (lymphatic nodes, spleen and thyroid gland), bone marrow, testicles, ovaries, gastrointestinal mucosa. Parenchymal organs, namely liver, adrenal glands, kidneys, salivary glands and lungs possess quite high radioresistance. According to World Health Organization (WHO), acute radiation sickness (ARS) is composed of the hematopoietic subsyndrome (HS), gastrointestinal subsyndrome (GIS), neurovascular subsyndrome (NVS) and cutaneous subsyndrome (CS) . The main factors which determine the survival of victims are the degree of bone marrow suppression and gastrointestinal tract damage due to the presence of a large pool of fast-dividing cells [20, 21, 22]. Acute radiation sickness (ARS) could be considered as a sequence of immediate radiation injury and long-lasting bystander cross-effects.
Management of patients with ARS includes early use of hematopoietic cytokines, antimicrobials and transfusion support; in addition, antiemetic agents and analgesics, and even hematopoietic stem cells transplantation [16, 23]. Since 1997, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are used and their doses are driven by the radiation dose and physiologic responses for ARS  and by clinical protocols for leukopenia and neutropenia caused by anti-cancer treatment [25, 26]. However, these drugs still are of high cost, and pharmacoeconomic benefits seem to be questionable . Singh et al. concluded that cytokine therapy has significant but modest effects . All these facts force the researches to search new methods and means for additions to the management of post-aggressive iatrogenic leukopenia and related ARS- and radiomimetic-induced damage.
3. Adsorptive hemoperfusion therapy for ARS
Sorption detoxification types, quite widely used today in medicine, are: (1) hemoperfusion (when blood is filtered through the column with activated carbon); (2) enterosorption—enteral use of oral adsorbents of a different type and (3) application-sorption therapy - use of carbon dressing for the healing of the burns and wounds.
The ground for use of direct perfusion of the blood through an adsorbent column for its purification (hemoperfusion) was the Kuzin A.M. Structural-Metabolic Theory in Radiobiology (1970) . Organs and tissues exposed to ionizing radiation and radiomimetic influences are damaged by radiotoxins, which affect radiosensitive structures, and direct radiation-dependent changes in the macromolecules of the genome. Further investigations demonstrated that “radiotoxins” are reactive oxygen species (ROS) formed by water radiolysis. Oxidative stress causes DNA, protein and lipid oxidation and is responsible for the whole range of signs and syndromes of ARS . Because of excessive lipid peroxidation, a lot of damaged cells appear that deepens the primary radiation injury repeatedly. In summary, ARS is a sum of primary damage due to oxidative stress plus so-called bystander effects , when cells exposed to ionizing radiation or radiomimetics can release signals that induce very similar effects on non-targeted neighboring cells.
Our first research of adsorptive therapy effects for acute radiation sickness (ARS) started in 1976 . In this study, 69 inbred dogs were irradiated by external X-ray at the dose of 525 Rad (5.25 Gy). They were randomly assigned to three groups: first control group (n = 31), which received standard antibiotics therapy; second group (n = 19) got antibiotics + hemoperfusion 2 hours after irradiation and third group (n = 19) underwent saline infusion 4–5 hours after irradiation plus furosemide, and hemoperfusion 24 hours later. The results are presented in Table 1.
|Group||Survival rate, %||Animals with critical hematological indices, %|
|Bone marrow cellularity <1.0 × 109/L||Leukopenia <1.0 × 109/L||Thrombocytopenia, <50.0 × 109/L|
|1 (n = 31)||3.2||13.4 ± 1.1||93.5||70|
|2 (n = 19)||68.4||17.0 ± 1.6||16.0||52.6|
|3 (n = 19)||62.4||16.6 ± 1.9||16.0||38.9|
The highest survival rate was in the second group—68.4%, while in the control group, it was only 3.2%. Late hemoperfusion also resulted in a high survival rate—62.4%. Only 16% of an animal with hemoperfusion treatment (three dogs in each group) had critical leukopenia. In the control group, it was 93.5% of animals.
It is noteworthy, that mitotic index (a marker of the rate of cells division) (Figure 1) was significantly higher in the second group compared to the control one (6 hours after irradiation) and even to the initial level (14th day after exposure to ionizing irradiation) .
Hemoperfusion with activated carbon also provided survival of 50% of dogs exposed to ionizing irradiation at the doses of 3.46 and 3.65 Gy . These results were re-tested and developed within a special closed program of Research institutions of the Ministry of Health and the Ministry of Defense of USSR. Hemoperfusion methods were implemented into clinics [33, 34].
A team of researchers who carried out the experiment on dogs by irradiating them at the dose of 5.25 Gy, witnesses that perfusions of the blood through the column with a carbon adsorbent were quite short. Slugging of columns was the main reason for incomplete procedures (only 0.3-0.5 of circulating blood volume was purified) . Despite these factors, the survival rate and other studied parameters were quite successful. We suppose that it could be explained by washout of dust particles from the surface of the adsorbent in the moment of primary contact with the blood, and viscosity changes inside the column after the replacement of rinsing solution to the blood also contributed to it. We think that positive secondary effects could be provided by nano- and microparticles (1–2 μ) of activated carbon, which contact with the blood. Their content is not controlled according to the standards of British (BP) and American (USP) Pharmacopeia.
Today, we have a lot of evidence that positive curative effects of carbon nanoparticles, alone or as a part of a composite, are obliged to their ability to scavenge the ROS and simulate suppose the effects of free oxygen radical scavenging enzymes. Sandhir R. et al.  believe that nanoantioxidants (inorganic nanoparticles possessing intrinsic antioxidant properties) would be more effective against ROS-induced damage because they cross the blood-brain barrier. It is a potential application in treating and preventing neurodegenerative conditions . Arifa R.D. et al. research demonstrated that nanocomposite with fullerol decreases the intensity of irinotecan-induced leukopenia and gastrointestinal damage in mice and do not diminish the tumoricidal effects of the drug . The aftertreatment with the same nanocomposite ameliorates the graft-versus-host disease reactions in mice and reduces intestinal lesions and bacterial translocation; prevents mortality and morbidity . Nano-fullerenes promote osteogenesis of human adipose-derived stem cells and possess a great antioxidant capacity .
Encouraging results have been found concerning the amelioration of side effects of one more radiomimetic—anthracycline antibiotic doxorubicin (DOX), which also is known by its ability to cause oxidative stress and leukopenia. Fullerenol C60(OH)24 nanoparticles improved the myocardial morphology of DOX-treated animals, but cause a certain degree of parenchymal degeneration by itself . Such and similar cases  evidence the need for designing and searching for the nanocomposites with specific features, which will possess antioxidant capacity without notable cytotoxicity. One of the solutions could be the conjugation of carbon nanomaterials with albumin . It was found that C60(OH)24 decreases the consequences of DOX-induced excessive oxidation in the tissues of kidneys, testis and lungs in mice . An aqueous solution of fullerenol was quite effective to fight experimental arthritis in rats . Andrievsky G.V. et al. demonstrated significant (but only by 15%) radioprotective properties of hydrate C60 fullerene in X-ray irradiation of the mice at the lethal dose of 7 Gy . Water-soluble polyvinilpyrroli done-wrapped fullerene derivative showed to significantly inhibit UVA-promoted melanogenesis in normal human epidermis melanocytes and human melanoma HMV-II cells within a non-cytotoxicity dose range . Huq R. et al. showed that nontoxic poly(ethylene glycol)-functionalized hydrophilic carbon clusters, known scavengers of the ROS superoxide and hydroxyl radical, are preferentially internalized by T lymphocytes over other splenic immune cells . It was successfully used to reduce T-lymphocyte-mediated inflammation in experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis) .
Another type of carbon material—carboxylated nanodiamonds, diminish the biochemical and histological signs of damage of γ-irradiated human erythrocytes . On the other hand, hydrogenated nanodiamonds dramatically increase the sensitivity to radiation effects of human radioresistant cancer cell lines . The same effect was seen considering the radiomimetic neocarcinostatin. Single-walled carbon nanotubes were found to be the efficient nanocarriers for drug delivery in the murine model of breast cancer [50, 51]. The team of researchers  synthesized the magnetic particles Fe3O4 in the shell from partially graphitized carbon and demonstrated their high intrinsic peroxidase-like catalytic activity, which promotes oxidative stress in human prostate cancer PC-3 cells in the presence of ascorbic acid. One more interesting study with a composite system of reduced graphene oxide—iron oxide nanoparticles showed that such a combination can synergistically induce physical and chemical damage to methicillin-resistant Staphylococcus aureus (MRSA) .
We must notice, that carbon nanoparticles possess great antioxidant properties and could be perspective for designing the nanopharmaceutical means and drugs to treat the disorders, when oxidative stress is an intrinsic part of pathogenesis, for leukopenia also. It means that further studies of carbon micro- and nanoparticles effects at parenteral routes of administration could finalize the discovery of quite a new method of mass treatment of acute radiation sickness.
Recently, several detailed reviews have been published on the pharmacological potential and prospects for the therapeutic use of cerium nanoparticles as traps of highly reactive oxygen (ROS) and nitrogen species (RNS) [54, 55, 56]. These reviews are based on a variety of experimental studies both in vitro and in vivo. Not less interesting results for use of nanocrystal cerium dioxide (CeO2) on the model of DOX-induced cardiomyopathy in rats we got . Cardiomyocytes mostly are damaged because of the radiomimetic impact of the drug, and the violation of blood components was quite similar to the effects of ionizing irradiation. It is known that oxidative stress is an intrinsic part of the cytotoxic effects of DOX, and heart tissues are vulnerable because of a lack of intracellular antioxidant defense factors compared to other organs and systems .
In this study, we used 21 female white mongrel rats, which were randomly assigned to the next groups (n = 7): first control groups got weekly intraperitoneal (IP) injection of saline; rats of second (DOX) and third groups got three times a week IP injections of doxorubicin at a dose of 2.5 mg/kg (n = 7); rats of third (DOX + CeO2) group got twice weekly IP injections of nanodisperse CeO2 (0.2 mg/kg) next day after doxorubicin injections additionally. Treatments lasted for 2 weeks (Figure 2).
Injections of nanodisperse CeO2 caused positive changes in myocardium structure. We observed improvement of a structure, decreased vacuolization of sarcoplasm, a number of cells with nuclei pathology was much lower (Figure 4) compared to the second group (Figure 3). A part of myocardium cells still had pyknotic nuclei with karyolysis signs. But mostly, the intensity of dystrophy and necrosis reduced and nuclei acquired oval shape again.
There were no significant positive changes in the structure of liver parenchyma. We may just note restoring nuclei sizes and shape and a little bit lighter pale pink color of cytoplasm. It witnesses that the synthetic function of the liver was partly restored. Concerning the kidneys, no positive changes had been found.
Biochemical indices of lipid and protein peroxidation, antioxidant defense system showed that CeO2 increased the activity of catalase by 24.6%, raised the level of reduced glutathione by 10.9% and decreased the level of oxidative modification of protein and lipids by 28.1 and 23.6%, respectively (compared to the group with untreated DOX-induced cardiomyopathy).
Bakht M.K. et al. proposed to reduce the actual radiation burden in patients exposed to radioisotope studies by arranging radiolabels for cerium oxide , and Colon J. et al. could achieve a good prophylactic result for radiation pneumonitis in mice that received nanocrystalline dioxide Ce . One more fact should be mentioned here: because of bone marrow suppression and leukopenia development, lungs are fragile to injury by ionizing irradiation. They have their own host defense system, based on alveolar macrophages. Because of leukocytes toxic damage (by ionizing injury or radiation therapy or as the side effects of anti-cancer chemotherapy), resting macrophages can no longer be transformed which lead to radiation pneumonitis . Heslet L. et al. showed that systemic administration of myelo stimulative cytokines was not helpful to prevent it because they do not penetrate the alveoli. That is why we suggest that oral adsorbents and/or parenteral use of CeO2 (it penetrated the alveoli and prevents radiation pneumonitis on mice model) will enhance the prophylaxis and treatment of ARS and decrease the intensity of side effects of radiation therapy and cytostatic drugs.
4. Local signs of whole-body irradiation and efficacy of application-sorption therapy
External exposure to ionizing irradiation frequently results in radiation burns of the skin. Leukopenia just deepens the injury because of oppressing the regeneration processes. A retrospective report on injuries caused by the atomic bombing of Hiroshima showed that up to 65% of all type of injuries were “radiation-combined injury,” when ionizing irradiation was coupled with burns, wounds and infections . Regarding these facts and negative contribution of leukopenia also, we want to demonstrate the efficiency of activated carbon. The remarkable result was observed on the model of the thermal non-full depth burn in Albino rats . The early application (within first 60 min) of the highly active carbon fabrics (SBET > 2000 m2/g) twofold reduced the healing time: 10.80 ± 1.27 and 20.60 ± 0.86 days for adsorptive carbon and gauze dressings use, respectively (Figure 8).
Similar results were observed on the burns caused by external irradiation at the dose of 8 Gy. Epithelialization of burn wounds has been completed on 21.1 ± 4.1 versus 27.3 ± 5.7 days after trauma for carbon and gauze dressings use, respectively. One more fact relates to the treatment ultraviolet radiation-induced burns. Application of adsorptive carbon dressings significantly (by 1.5–1.7 times) accelerated the burn-healing time. All these data will be published soon.
These results presented the undoubted perspective for use of high capacity carbon fabrics for the treatment of superficial skin lesions, especially complicated by concomitant leukopenia.
5. Enterosorption for leukopenia management
Hemoperfusion as a procedure requires well-trained staff, specific equipment and sterility. It means that such method of sorption detoxification is not adapted to emergency exposure situations, during war-time and large human contingent injury. That is why the use of enteral sorption therapy (ingestion of activated carbon) is a more prospective method for such situations. Among the early studies, the great results were observed in the patients with lymphogranulomatosis undergoing radiotherapy , who were treated with fibrous carbon oral adsorbent. Enterosorption treatment allowed to continue planned schemes of radiation therapy and was more efficient than conventional methods for leukopenia healing. In the next study , cyclophosphane was given to Guerin tumor-grafted rats at the dose of 100 mg/kg of body weight on 10th and 13th days after tumor transplantation; enterosorption with synthetic SCN carbons (bulk density 0.3–0.4 g/cm3) was administered next day after cyclophosphane injection. These expressed myeloprotective effects we approved and confirmed in the clinic. One more radiomimetic anti-cancer agent cisplatin was used in an experiment on Guerin tumor-grafted rats  and highly activated fibrous carbon material Carboline (Ukraine) successfully ameliorated a wide range of its side effects. Carboline is used in clinical practice also and demonstrates promising results .
Our latest experiments on rats exposed to X-ray irradiation in a total dose of 6 Gy (63 Rad per min, t = 11 min) demonstrated great results of novel oral carbon adsorbents administration to ameliorate radiation-caused leukopenia. We used two granulated activated carbons (AC) with a diameter of granules (0.25–0.5 mm) and bulk density 0.1 and 0.2 g/cm3 (ES1 and ES2, respectively). Enterosorbents were administrated as radioprotectors, radiomitigators and therapeutic agents (at the dose of 10 ml/kg, admixed to the food, three days before and nine days after ionizing irradiation exposure). Irradiation caused a 10-fold decrease in the white blood cells count. ES1 administration raised the index twice on the 9th day after X-ray exposure, while ES2 produced fewer results (Figure 12).
The same effect was observed concerning the lymphocytes count (Figure 13). Structural differences among those two carbon adsorbents are estimated. These results will be published soon in detail.
So, as we observed, specific oral adsorbents with specified porosity and pores distribution are quite successful to fight the iatrogenic leukopenia because of the influence of ARS or anti-cancer treatment.
Oral carbon materials have a high capability to decrease the emesis caused by anti-cancer treatment [66, 67]. Also, it is a unique mean with anti-diarrhea action, which could be implemented in the clinics for the treatment of ARS-induced gastrointestinal subsyndrome as well as for dyspepsia syndrome caused by tumoricidal therapy.
Thus, enterosorption for the results on the animal study and use in clinics do prevent hematotoxicity of anti-cancer treatment and significantly ameliorated leukopenia and its consequences.
6. Oral carbon adsorbents as an addition to classical treatment of leukopenia with G-CSF
Abovementioned positive results led us to design of a new improved version of carbon oral adsorbent, which was used in early experiments [64, 68]. An old prototype C1 and his new version C2 were approved on the model of melphalan-induced bone marrow suppression [69, 70, 71]. We demonstrated that myeloprotective action of carbon granulated enterosorbent С1 (bulk density of 0.28 g/cm3, specific surface of 1719 m2/g and mesopore area of 239 m2/g) is significantly less compared to effects of adsorbent C2 with bulk density of 0.18 g/cm3, total specific surface of 2162 m2/g and mesopore area of 565 m2/g (Figure 14).
C2 enterosorbent administration normalized the prooxidant/antioxidant system indices too . Oxidative stress is an intrinsic part of ionizing radiation and radiomimetic injury, and enteral sorption therapy possesses notable antioxidant effects.
Adding the carbon oral adsorbent to classical scheme for the treatment of leukopenia with G-CSF (caused by single intravenous melphalan injection at the dose of 4 mg/kg) demonstrated significant myeloprotective effect and synergy compared to single-use effects of each agent alone  (Figures 15 and 16). C2 and filgrastim combination caused increase of white blood cells count by 138.3% compared to the melphalan group; by 65.8% compared to melphalan + C2 group, and by 51.7% compared to use of filgrastim alone.
We must note that in this study, we got an unexpected significant increase of platelets level from (254.60 ± 45.59) to (505.40 ± 70.68) × 109/L in a group of rats that received the combined treatment with oral adsorbent and G-CSF. Isolated administration of enterosorbent C2 tended to raise the level of thrombocytes, we suppose because of general detoxification action.
Use of enterosorbent or combined use of both preparations provided significantly better effects toward the prooxidant/antioxidant balance in rats.
The important issue is that combination of G-CSF and carbon adsorbents  as well as enteral sorption therapy use alone  does not affect the efficacy of anti-cancer treatment; we proved it by our experiments on Guerin tumor-grafted rats.
Leukopenia is an essential part of the damage caused by ionizing irradiation and/or radiomimetic influences (as tumoricidal chemotherapy). Leukocytes play an important role in immune defense, tissue regeneration, the functioning of the main organs and systems; and the degree of bone marrow suppression determines the survival of victims in ionizing radiation exposure as well as the efficacy of anti-cancer chemotherapy. All three methods of sorption detoxification with activated carbons such as hemoperfusion (when blood is filtered through the column with activated carbon); enterosorption—peroral use of oral adsorbents and application-sorption therapy (use of carbon dressing for the healing of the burns and wounds), can be successfully used for the leukopenia prophylaxis and treatment in ionizing irradiation exposure, side effects of anti-cancer chemotherapy, as well as for the boosting of healing of associated skin damage.
Enterosorption demonstrates significant synergy with hemopoietic cytokines in the treatment of bone marrow suppression caused by such an aggressive agent as melphalan (a derivative of mustard nitrogen). Nanocrystal cerium dioxide could be useful for oxidative stress modulation caused by such radiomimetic as anti-cancer anthracycline antibiotic doxorubicin. Modification of pathological biochemical processes which provoke bone marrow suppression and leukopenia is a basis of the efficacy of sorption detoxification in acute radiation syndrome as well as to decrease the side effects of anti-cancer chemotherapy. Our findings may contribute to the refinement of current risk stratification algorithms for acute radiation sickness treatment.
Conflict of interest
The authors declare the absence of the conflict of interest.
|NPP||nuclear power plants|
|ARS||acute radiation sickness.|
|G-CSF||granulocyte colony-stimulating factor.|
|ROS||reactive oxygen species.|
|Gy||gray, unit of ionizing radiation dose.|