Study of the Mechanisms Underlying the Development of Anemia during Subacute Polychlorinated Biphenyl Intoxication (Experimental Study)

The dissertation presents an experimental study of the mechanisms underlying hemolytic hypochromic anemia in rats during subacute intoxication with various doses of polychlorinated biphenyls. It covers the effects of PCBs on the erythron, iron and porphyrin metabolism, erythropoiesis, splenic and bone marrow macrophage function, and lipid peroxidation and antioxidant defense.

The study used a systematic approach and pathophysiological, biochemical, and morphological methods; rats were administered PCBs at doses of 0.5, 0.05, and 0.025 LD50. Peripheral blood indicators, iron and porphyrin metabolism, luminol-dependent chemiluminescence, and the state of the erythron were assessed. The findings indicate an association between hematologic abnormalities and accelerated erythrocyte destruction followed by enhanced erythrocyte formation in bone marrow, with the nature of the changes depending on PCB dose.

Among the environmental hazards threatening humanity at present, the foremost role is attributed to anthropogenic chemical pollution of the environment. An enormous number of substances that are foreign to living organisms (xenobiotics) enter the biosphere together with products or waste. Particular attention is warranted for the group of persistent organic pollutants (POPs), which share a number of common properties. All of these substances belong to the class of chlorinated organic compounds; they are resistant to degradation, circulate in the natural environment for long periods, accumulate in living tissues, and can be detected even in places where industry does not exist (Bobovnikova Ts. I., 1982; Gusakov A. E., 1992; Kayumova A. F., 1996; Maistrenko V. A., 1998; Voronov V. V., 1999; Klyuev N. A., 2001; Dachs J., 2002).

Polychlorinated biphenyls (PCBs) are classified as organochlorine compounds that are super-ecotoxicants, spreading far beyond their original sites of occurrence and, even at trace levels, exerting a detrimental effect associated with damage to cell membranes and disruption of all types of metabolism in humans and animals (Benes V., 1980; Revich B. A., 2002, 2006; Singh A., 1997; Dachs J., 2002).

PCBs are widely used in human industrial activity. These sectors include ferrous metallurgy, energy, forestry, and agriculture. Accumulated stocks of biphenyls in the biosphere, even during a gradual slowdown in their production, do not make it possible to eliminate the resulting problem (Dachs J., Lohmann R., Ockenden W. A., et al., 2002), because PCBs are highly persistent in the environment. They do not undergo hydrolysis, oxidation, or biodegradation. PCBs enter the environment primarily through the discharge of industrial wastewater into rivers, where they accumulate in silty sediments, which can subsequently be used as fertilizer. PCBs enter the external environment predominantly in vapor form (Benes V., 1980; Dachs J., 2002).

PCBs accumulate in foods such as milk, cereals, and greens, as well as in animals and fish, primarily in their adipose tissue (Kryzhanovskaya M. V., 1993). With subacute and chronic exposure to PCBs in humans and animals, a complex of pathological symptoms develops. These include progressively worsening hepatic failure, hyperkeratosis, skin pigmentation, and lesions of the endocrine, nervous, and reproductive systems (Lashneva N. V., 1989; Abdullina G. M., 1999; Galimov Sh. N., 2000; Mufazalova N. A., 2004; Myshkin V. A., 2006; Khabirov R. E., 2006). Experimental studies have demonstrated the embryotoxic, teratogenic, mutagenic, and immunosuppressive effects of PCBs (Tutelyan V. A., 1986; Volkov V. S., 1998; Volkova E. S., 2000; Basyrova N. K., 2001; Sabirova I. R., 2004).

Thus, studying the response of the human and animal organism to PCB intoxication is one of the urgent medical and biological problems of the present day, because the environmental situation in the country and worldwide has not improved (Kryzhanovskaya M. N., 1993; Tutelyan V. A., Lashneva N. V., et al., 1995; Maistrenko V. N., 1998; Voronov V. V., 1999).

The biological action of PCBs is multifaceted, manifested in toxic effects in various tissues. It is currently established that biphenyls affect lipid peroxidation in cell membrane lipids, which is expressed through the initiation and enhancement of lipid peroxidation (LPO) (Tutelyan V. A., et al., 1986; Volkov V. S., 1998; Basyrova N. K., 2001; Shaikhullina L. R., 2004).

A. F. Gaysina (2000–2002) and A. V. Bogdanova (2004–2006) demonstrated that PCBs cause hemolytic anemia in experimental animals, characterized by the formation of erythrocytes with low hemoglobin content. However, the mechanisms underlying the development of hypochromic anemia during intoxication of experimental rats with various doses of PCBs have not been elucidated: iron metabolism, porphyrin metabolism, and the functional state of splenic and bone marrow macrophages during intoxication of experimental rats with various doses of PCBs have not been investigated.

For this reason, the nature of the anemia accompanying PCB intoxication was investigated according to a systematic approach using pathophysiological, biochemical, and morphological methods. The state of the prooxidant-antioxidant system was assessed, including antioxidant defense enzymes—superoxide dismutase and catalase—products of lipid peroxidation in erythrocytes, bone marrow, and liver, antioxidant activity of blood serum and liver, luminol-dependent chemiluminescence in erythrocytes, blood serum, bone marrow, and liver, indicators of iron and porphyrin metabolism, and the peripheral and central compartments of the erythron in experimental rats exposed to PCBs at doses of 0.5 LD50, 0.05 LD50, and 0.025 LD50.

Study Objective

The objective of the study was to investigate the mechanisms underlying the development of hemolytic hypochromic anemia in rats exposed to various doses of PCBs by examining iron and porphyrin metabolism, erythropoiesis in erythroblastic islands of bone marrow, the functional state of splenic and bone marrow macrophages, changes in lipid peroxidation processes (LPO), and antioxidant activity (AOA) in erythrocytes, blood serum, bone marrow, and liver.

Study Objectives

1. To examine indicators of peripheral blood (erythrocyte and hemoglobin content, reticulocyte count, red blood cell indices, and hematocrit) during subacute PCB intoxication at doses of 0.5 LD50, 0.05 LD50, and 0.025 LD50.

2. To examine the pattern of iron metabolism (serum iron ion concentration, total iron-binding capacity, unsaturated iron-binding capacity, percentage of transferrin saturation with iron, serum ferritin level, amount of free hemoglobin in blood serum, presence of hemosiderin in 24-hour urine, and deposition of hemosiderin in cells of the spleen and liver) in experimental animals during subacute PCB intoxication at doses of 0.5 LD50, 0.05 LD50, and 0.025 LD50.

3. To study indicators of porphyrin metabolism (coproporphyrin and protoporphyrin content in erythrocytes, coproporphyrin and 8-aminolevulinic acid content in 24-hour urine, and total bilirubin concentration in blood serum) during subacute experimental animal intoxication with PCBs at doses of 0.5 LD50, 0.05 LD50, and 0.025 LD50.

4. To assess the functional state of the system of phagocytizing mononuclear cells, in particular, splenic macrophages and central macrophages of bone marrow, during PCB intoxication of rats with doses of 0.5 LD50, 0.05 LD50, and 0.025 LD50.

5. To examine the state of the prooxidant-antioxidant system in blood serum, erythrocyte membranes, bone marrow, and liver during the administration of PCBs at doses of 0.5 LD50, 0.05 LD50, and 0.025 LD50.

6. To examine luminol-dependent chemiluminescence in blood serum, erythrocytes, and bone marrow in rats exposed to PCBs at doses of 0.5 LD50, 0.05 LD50, and 0.025 LD50.

Scientific Novelty

The data obtained made it possible to establish the nature of disturbances in iron and porphyrin metabolism and the functional state of splenic macrophages and central bone marrow macrophages in experimental rats exposed to PCBs at doses of 0.5 LD50, 0.05 LD50, and 0.025 LD50. Studies of lipid peroxidation and chemiluminescence in bone marrow and of the activity of antioxidant defense enzymes were conducted for the first time. Increases in intermediate and final LPO products, decreases in antioxidant enzyme activity in bone marrow and liver, and decreases in the overall antioxidant activity of blood serum were identified. It was demonstrated that hematologic abnormalities in animals exposed to PCBs, characterized by accelerated erythrocyte destruction followed by enhanced erythrocyte formation in bone marrow, are dose-dependent.

The data obtained made it possible to assess the severity of the hematologic syndrome, identify the pathogenesis of anemia during PCB intoxication, and assess the possibility of developing methods for preventing hematologic manifestations associated with exposure to various doses of PCBs.

Practical Significance

Experimental confirmation was obtained of the development of hypochromic anemia in rats during intoxication with various doses of PCBs. The data obtained provide a basis for developing diagnostic and preventive algorithms for various disorders of the erythroid blood system and liver during the development of toxic hepatitis in individuals directly involved in the production and use of PCBs, as well as in residents of environmentally unfavorable areas.

Theses for Protection

1. Intoxication of experimental animals with various doses of PCBs causes hypochromic hemolytic anemia of the regenerative type, which is replaced by the end of the experiment (day 28) by hemolytic hypochromic anemia of the hyporegenerative type.

2. Rats exposed to PCBs at doses of 0.5 LD50 and 0.05 LD50 exhibited more intense erythrocyte hemolysis, characterized by increases in serum iron ion concentration, transferrin saturation with iron, serum ferritin, and free hemoglobin by the end of the experiment. Erythrocyte hemolysis was less pronounced in the 0.025 LD50 group.

3. In rats exposed to PCBs at doses of 0.5 LD50 and 0.025 LD50, suppression of porphyrin metabolism was more pronounced and consisted of increased coproporphyrin and protoporphyrin content in erythrocytes and increased 8-aminolevulinic acid content in 24-hour urine. These changes were less pronounced in the 0.05 LD50 group.

4. Examination of the functional activity of the phagocytizing mononuclear cell system revealed increased activity in the groups of rats exposed to the toxin at doses of 0.5 LD50 and 0.025 LD50, characterized by greater iron uptake by splenic macrophages than in the 0.05 LD50 group.

5. Erythrocyte maturation in bone marrow was uneven in all groups, as manifested by delayed maturation of erythroid cells in erythroblastic islands. However, the partial recovery of erythrocyte count and reticulocytosis at the end of the experiment in the 0.025 LD50 group indicates preserved compensatory capacity of the central erythron in these animals.

6. All experimental groups exhibited enhanced lipid peroxidation in the liver and bone marrow and suppression of the antioxidant system in erythrocytes and blood serum.

7. The study of luminol-dependent chemiluminescence revealed enhanced LPO in bone marrow and erythrocytes in all groups of experimental animals from the first day after the experiment began.

Presentation of the Dissertation Results

The results were presented at:

- 66th Republican Final Scientific Conference of Students and Young Scientists of Bashkir State Medical University with International Participation. — Ufa, 2001;

- 68th Republican Final Scientific Conference of Students and Young Scientists of Bashkir State Medical University with International Participation. — Ufa, 2003;

- Republican Conference of Young Scientists of the Republic of Bashkortostan “Medical Science—2003”. — Ufa, 2003;

- Republican Conference of Young Scientists of the Republic of Bashkortostan “Scientific Breakthrough—2003”. — Ufa, 2003.

- 69th Republican Final Scientific Conference of Students and Young Scientists of Bashkir State Medical University with International Participation. — Ufa, 2004;

- IX All-Russian Scientific and Practical Conference “Young Scientists in Medicine—2004”. — Kazan, 2004.

- Interregional Scientific and Practical Conference with International Participation “Typical Pathological Processes”. — Ufa, 2005.

- 71st Republican Final Scientific Conference of Students and Young Scientists of Bashkir State Medical University with International Participation. — Ufa, 2006.

- Republican Scientific and Practical Anniversary Conference “Relevant Issues of Contemporary Medicine and Healthcare”, Dedicated to the 70th Anniversary of the Department of Public Health and Organization of Healthcare of Bashkir State Medical University. — Ufa, 2006.

- International Symposium “Relevant Problems of Adaptation in Northern Conditions”. — Khanty-Mansiysk, 2006.

- 1st International Scientific Conference “Adaptation of Biological Systems to Natural and Extreme Environmental Factors”. — Chelyabinsk, 2006.

- 3rd Congress of Urals Physiologists. — Yekaterinburg, 2006.

- X All-Russian Medical and Biological Conference of Young Researchers. — St. Petersburg, 2007.

According to the dissertation results, 15 printed publications were published. The study was conducted at the Department of Normal Physiology of Bashkir State Medical University (headed by Doctor of Medical Sciences, Professor A. F. Kayumova; rector: Corresponding Member of the Russian Academy of Medical Sciences, Doctor of Medical Sciences, Professor V. M. Timerbulatov).

Volume and Structure of the Dissertation

The dissertation consists of an introduction, a literature review, a chapter entitled “Materials and Methods of the Study”, a chapter entitled “Original Data”, a discussion of the results obtained, conclusions, practical recommendations, and a list of references.