Blood and Bone Marrow Toxicity Biomarkers

C H A P T E R

23 Blood and Bone Marrow Toxicity Biomarkers Sharon Gwaltney-Brant Veterinary Information Network, Mahomet, IL, United States

INTRODUCTION The blood and bone marrow are vital to survival, providing oxygen and nutrients to tissues, protecting the host from exogenous and endogenous invaders, providing the conduit for multisystemic communications, and preventing undue loss of bodily fluids. Because of its widespread influence within the body, toxic insults to the bone marrow or its daughter cells can result in severe consequences such as hypoxia, overwhelming infections, malignant neoplasia, or hemorrhage. The hematopoietic system contains the most mitotically active cells in the body, making it a prime target for toxicants that attack rapidly dividing cells. The ability to quickly and effectively detect and measure toxicant-induced injury to hematopoietic or mature blood cells may allow for the institution of measures to mitigate the damage caused by the toxicant. Utilizing biomarkers as tools to predict the potential risks to the hematopoietic system posed by xenobiotics can enable us to preferentially select those compounds showing the least adverse effects on the body.

HEMATOPOIETIC SYSTEM The hematopoietic system is composed of the bone marrow and the various cells that it produces. The production of blood cells is highly regimented and complex, involving innumerable cytokines, chemokines, neuropeptides, enzymes, and other chemical mediators. Although much has been learned about the intricate interactions among these players, there is still much that is not known about the mechanisms of hematopoietic cell proliferation, differentiation, maturation, and function.

Bone Marrow In adults, hematopoietic marrow is concentrated in the spine, pelvis, sternum, ribs, calvarium, and Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00023-2

proximal ends of the limb bones (Valli, 2007). Tucked away in the protective casing of cortical and cancellous bone, marrow is composed of hematopoietic cells, adipose tissue, and adjacent supportive cells and tissues. The microenvironment produced by the unique endosteal blood flow patterns, including boneebone marrow portal capillary systems, provides the appropriate milieu for the proliferation, differentiation, and maturation of cellular components of the blood. Stromal stem cells give rise to adipocytes, osteoblasts, chondroblasts, and reticular cells that produce the structural scaffolding of the marrow and that secrete soluble mediators essential for the maintenance, differentiation, and growth of hematopoietic stem cells. In immature and young animals bone marrow is red, reflecting the high hematopoietic activity (Stockham and Scott, 2008). With age, red marrow is replaced by yellow marrow, which is composed of adipose tissue that differs from fat of other body sites in that marrow fat is more resistant to lipolysis in response to starvation (Valli, 2007). Conversion of yellow marrow back to red marrow may occur associated with pathologic states that stimulate hematopoiesis (e.g., anemia) and is most common in areas with higher blood flow such as endosteal surfaces.

Hematopoiesis Hematopoiesis in the fetus occurs in multiple organs besides the bone marrow, including the thymic anlage, primordial lymph nodes, liver, spleen, kidney, and adrenals (Valli, 2007). At birth hematopoiesis is restricted primarily to the marrow, although occasional areas of extramedullary hematopoiesis may be found in the spleen or liver. In adults, extramedullary hematopoiesis (EMH) may occur in association with hematological disorders when bone marrow hematopoiesis is insufficient or ineffective. Typical sites of EMH include liver, spleen, lymph nodes, and paravertebral areas with the

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23. BLOOD AND BONE MARROW TOXICITY BIOMARKERS

particular cell type (e.g., erythrocytes in an anemic patient), production of other cell lines (e.g., neutrophils) will be reduced (Valli, 2007). Ontogenic relationships can also influence the relative production of cell lines; for example, erythroid and megakaryocytic cells derive from the same precursor cell lineage, and stimuli that increase production of erythrocytes frequently result in concurrent increases in platelet numbers. As an understanding of differentiation, growth, and kinetics of the cells and soluble mediators is essential to interpretation of toxicant-induced bone marrow and blood cell injury, a brief description of the myeloid cell lines follows.

intraspinal canal, presacral region, nasopharynx, and paranasal sinuses being less common locations for EMH (Sohawon et al., 2012). Although hematopoietic stem and progenitor cells capable of producing hematopoietic cells in vitro have been found in the stromal vascular fraction of adult adipose tissue, spontaneous EMH in adipose tissue is rare (Han et al., 2010).

Hematopoietic Cells Hematopoiesis begins with the pluripotential stem cell, a primitive cell with almost unlimited capacity for self-renewal and the ability to differentiate into any of the blood cell lines. Growth factors and interleukins within the marrow microenvironment orchestrate the growth and differentiation of stem cells. With each level of differentiation, the commitment of the cell to a single cell type becomes more and more entrenched (See Fig. 23.1). In the event of an unusual demand for one

Erythrocytes Erythrocytes comprise up to 45% of the circulating blood volume and are vital to the transport of oxygen from the lungs to the tissues, as well as the transfer of carbon dioxide from the tissues to the lungs for Pluripotential stem cell SCF, IL-6, Flt3L

CFU-GEMM

Lymphoid stem cell SCF, Flt3L, Il-7

IL-3, GM-CSF, IL-6

TPo

GM-CSF

IL-11

Epo

Pre-B cell

CFU-GM

CFU-E-MK

IL-3

TPo

BFU-E

Megakaryocyte

CFU-B

Erythrocyte

Platelet

Basophil

IL-5

M-CSF

CFU-Eo

CFU-M

Eosinophil

Monocyte

G-CSF

IL-6

CFU-N

Neutrophil

Flt3L

CFU-MC

Mast cell

B Cell

Pre-T cell

IL-7

T Cell

Plasma cell

FIGURE 23.1 Differentiation of mammalian hematopoietic cells. Boxes contain primary soluble mediators for each cell lineage. BFU-E, blast forming unit, erythrocyte; CFU-B, colony forming unit, basophil; CFU-E-MK, colony forming unit erythrocyte, megakaryocyte; CFU-Eo, colony forming unit, eosinophil; CFU-GEMM, colony forming unit granulocyte, erythrocyte, macrophage, megakaryocyte; CFU-GM, colony forming unit granulocyte; CFU-M, colony forming unit, monocyte; CFU-MC, colony forming unit, mast cell; SCF, stem cell factor; CFU-N, colony forming unit, neutrophil; Epo, erythropoietin; Flt3L, Flt3 ligand; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colonystimulating factor; IL, interleukin; M-CSF, monocyte/macrophage colony-stimulating factor; TPo, thrombopoietin.

II. SYSTEMS TOXICITY BIOMARKERS

HEMATOPOIETIC SYSTEM

TABLE 23.1

Species Variation in Life Spans of Erythrocytes

403

inflammatory cells via soluble mediators. Platelets are not cells, but rather cytoplasmic fragments derived from megakaryocytes. Megakaryocytes are formed from the erythroid/megakaryocytic precursor cell under the continued influence of thrombopoietin. It takes about 4 days for platelets to be produced and, once released into the blood, platelets have a life span of 5e10 days (Valli, 2007).

Species

Average RBC Life Span (days)

Cat

70a

Cattle

150a

Dog

100a

Horse

150a

Human

120a

Rabbit

55b

Monocytes

Rat

58b

Monocytes account for w5% of the total circulating leukocyte population and they are the precursors to tissue macrophages (Tizard, 2013). As part of the innate immune system macrophages function as phagocytes, removing pathogens and necrotic debris from sites of inflammation. Acting as antigen-presenting cells, macrophages also function in the acquired (adaptive) immune response. Under stimulation by IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and finally macrophage colony-stimulating factor, the myeloid stem cell gives rise to the monocyte precursor and ultimately differentiates into the monocyte. Once released into the bloodstream, monocytes circulate for approximately 3 days before entering tissues where they can replicate or differentiate into macrophages (Tizard, 2013). Tissue macrophages have extremely variable life spans, but generally are considered longlived cells.

a

Stockham and Scott (2008). Rodnan et al. (1957).

b

exhalation (Bloom and Brandt, 2008). Erythrocytes are also important in maintaining homeostatic blood pH, clearing of immune complexes and complement fragments, and regulation of blood flow to tissues. Stimulation of the common myeloid stem cell by interleukin-3 (IL-3), IL-6, IL-11, granulocyte colony-stimulating factor, and thrombopoietin results in the differentiation of the myeloid stem cell into the erythroid/megakaryocytic precursor cell (Aster, 2005). In the presence of erythropoietin, which is produced in the fetal liver and adult kidney, further differentiation into an erythrocyte colony forming unit (CFU) occurs, resulting in the development of erythroblast, which synthesizes and accumulates hemoglobin and eventually extrudes the nucleus, forming a reticulocyte. Reticulocytes retain stainable RNA and ribosomes which give the cells their name; as the RNA and ribosomes are lost, the reticulocytes become mature erythrocytes and are released into the circulation. Erythropoiesis requires approximately 4 days to complete. Toxicants that interfere with cell differentiation, proliferation, or growth can alter erythrocyte production, resulting in anemia or polycythemia. Toxicants that alter hemoglobin synthesis (e.g., lead) result in erythrocytes with reduced oxygen-carrying capacity and increased membrane fragility (Thompson, 2018). Erythrocyte life spans vary with species (Table 23.1) and are related to the level of oxygen-derived radicals formed and the efficiency of the intrinsic erythrocyte antioxidant systems for each species (Kurata et al., 1993). Toxicants that deplete or damage erythrocyte antioxidant systems or increase free radical formation can shorten the erythrocyte life span.

Platelets Platelets function in the formation of the hemostatic plug to mitigate hemorrhage from vascular damage. Platelets also play roles in wound healing and inflammation, primarily through communication with

Neutrophils The neutrophil is the most abundant leukocyte in circulation, comprising up to 75% of circulating white blood cells; up to two-thirds of the hematopoietic output of the bone marrow is composed of neutrophils (Tizard, 2013). Neutrophils are the first line of defense in innate immunity as they act as phagocytes and also have direct killing capabilities. Neutrophils share a common precursor cell with monocytes and differentiate under the influence of granulocyte stimulating factor. Neutrophils take approximately 6 days to be produced and have a relatively short life span of less than 12 h after entering the blood.

Eosinophils Eosinophils have phagocytic and bactericidal capabilities, play a role in immune defense against parasites, and inactivate mediators released from mast cells (Stockham and Scott, 2008). Eosinophil precursors differentiate from myeloid precursor cells stimulated by IL-3, IL-5, IL-6, and granulocyte-macrophage colony-stimulating factor; further IL-5 exposure

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23. BLOOD AND BONE MARROW TOXICITY BIOMARKERS

stimulates their differentiation into eosinophiloblasts which then mature into eosinophils. After minutes to hours in the blood, eosinophils migrate into tissues where they may persist for up to a few weeks.

Basophils

factors, and interleukins (especially IL-3, IL-5, IL-6, IL7, IL-11, and IL-15) to stimulate proliferation and mediate the commitment of cells to their respective lineages (Lyman, 1995).

MECHANISMS OF HEMATOTOXICITY

Basophils are the least abundant granulocyte population as they account for less than 1% of circulating leukocytes. Basophils play a role in immediate hypersensitivity disorders, as well as atopy, allergic contact dermatitis, and possibly autoimmune diseases such as systemic lupus erythematosus (Siracusa et al., 2011). Basophils can act as antigen-presenting cells, and they have antiparasitic functions similar to eosinophils. Basophils differentiate from myeloid stem cells under the influence primarily of IL-3 and other, as yet unidentified cytokines. Once mature, basophils have an estimated life span of 60e70 h (Siracusa et al., 2011).

Lymphocytes Circulating lymphocytes account for 20%e35% of the leukocyte population. Lymphocytes differentiate from the common lymphoid precursor cell under the influence of IL-7, Flt3 ligand, and stem cell factor; further exposure to IL-7 stimulates maturation of T cells within the thymus, whereas Flt3 ligand stimulates B cell maturation in the bone marrow. Refer to Chapter 24, “Immunotoxicity Biomarkers,” in this book for more detailed descriptions of lymphoid cells.

Soluble Mediators A large number of soluble mediators have been identified that exert tremendous influence in the proliferation, differentiation, growth, maturation, and function of cells of the bone marrow and blood (Fig. 23.1). These include growth factors, colony-stimulating factors, interleukins, chemokines, and neuropeptides. Stem cell factor, an activator of the receptor tyrosin kinase c-Kit, is crucial for the initiation of normal hematopoiesis and, along with IL-6 and Flt3 ligand, mediates the commitment of pluripotential cells to lymphoid or myeloid lineages (Lennartsson and Ronnstrand, 2012). Flt3 ligand is another hematopoietic cytokine that interacts with tyrosine kinase III receptors essential for the initiation, expansion, and maintenance of hematopoiesis (Wodnar-Filipowicz, 2003). Expression of Flt3 receptors is limited to hematopoietic cells lacking lineagespecific markers, so they are found on the most primitive (i.e., least differentiated) hematopoietic cells. Stem cell factor and Flt3 ligand act synergistically with a wide range of specific colony-stimulating factors, growth

With so many cells, mediators, and enzymes involved in the production and maintenance of the bone marrow and blood, there are countless potential mechanisms by which toxicants can exert their adverse effects. Within the hematopoietic stem cell population only a fraction of cells is undergoing replication, with the majority of cells in a resting phase that protects them from toxic insult from external forces that target rapidly dividing cells (e.g., ionizing radiation, chemotherapeutic agents). Some toxicants such as lindane are directly cytotoxic to hematopoietic progenitor cells regardless of the phase of cell cycle, causing necrosis of cells with subsequent myelosuppression (Parent-Massin et al., 1994). Direct damage to mature cells by toxicants can lead to hemolytic anemia, thrombocytopathy, or depressed immune function due to depletion of erythrocytes, platelets, or leukocytes, respectively. (Rebar, 1993) Indirect injury to blood or bone marrow cells can occur through a variety of means, including stimulation of immune responses against cell membranes or other structures, interference with cell surface receptors preventing normal cell function, and interference with cytokines or other soluble mediators necessary for hematopoietic cell proliferation, differentiation, maturation, or maintenance. For instance, the antineoplastic paclitaxel has been shown to alter the bone marrow microenvironment, which decreases the sensitivity of late erythroid progenitors to erythropoietin, resulting in depletion of erythroid precursors during late erythropoiesis (Juanisti et al., 2001). Toxicants may interfere with enzyme systems essential for normal cell function by inhibition of enzymes or interference with cofactors, such as occurs with interference with folate or cyanocobalamin by ethanol (Bloom and Brandt, 2008), and with decreased erythrocyte d-aminolevulinic acid dehydrogenase activity in lead toxicosis (Jangid et al., 2012; Feska et al., 2012). Toxicant-induced alteration of enzymes involved in hemoglobin synthesis can result in mature erythrocytes with reduced oxygen-carrying capacity due to deficient hemoglobin levels. Platelet function can be permanently disabled by inhibitors of cyclooxygenase such as aspirin (Hall and Mazer, 2011). Inhibition of vitamin K epoxide reductase by coumadin-based anticoagulants (e.g., warfarin) results in depletion of vitamin K-dependent clotting factors II, VI, IX, and X, resulting in coagulopathy. Mature erythrocytes with normal hemoglobin levels can have their oxygen-carrying capacity

II. SYSTEMS TOXICITY BIOMARKERS

MARKERS OF HEMATOPOIETIC/HEMATOLOGIC TOXICITY

altered by toxicants such as carbon monoxide or methemoglobin-inducing agents (e.g., nitrites).

BIOMARKERS OF HEMATOTOXICITY A variety of assays are used clinically and nonclinically to evaluate the status of the bone marrow and blood (Table 23.2). Although newer technologies such as flow cytometry and automated cell counters have improved the speed and efficiency of some of the benchmark assays, microscopic examination of these tissues is still necessary for full evaluation of bone marrow and blood cell status. Biomarkers of leukocyte toxicity are discussed in Chapter 22, “Immunotoxicity Biomarkers.”

MARKERS OF HEMATOPOIETIC/ HEMATOLOGIC TOXICITY Complete Blood Count The complete blood count (CBC) is the easiest, quickest, and most cost-effective method to get a rapid snapshot of the status of the blood and bone marrow, making it the most efficient means of screening the blood for evidence of toxicant-related injury (Adewoyin and Nwogoh, 2014). At a minimum, the CBC should include the following parameters: hematocrit (also called packed cell volume; Hct or PCV), mean cell volume (MCV), mean cell hemoglobin concentration (MCHC), total white blood cell count (WBC), differential white blood cell count, platelet count, and evaluation of stained blood smears. Although modern blood analyzers can perform the cell counts, manual evaluation of the stained blood smear is necessary to verify accuracy of the counts when pathologic conditions are present. For example, nucleated red blood cells present in the circulation of lead poisoning cases may be read out by the machine as white blood cells. Similarly, morphologic aberrations due to toxicants, such as the presence of Heinz bodies in oxidant-induced hemolytic anemias or basophilic stippling in lead toxicosis, will need to be evaluated visually (Table 23.3). Platelet clumping may result in falsely lowered manual and automated platelet counts, again emphasizing the need for light microscopic confirmation. Abnormalities in hematocrit can include polycythemia, such as is seen with cobalt (Simonsen et al., 2012). Anemia is the more common hematocrit abnormality and may be the result of increased erythrocyte loss, such as in hemolytic anemia induced by oxidants, or decreased production due to bone marrow suppression. Mean cell volume is a measure of the size of erythrocytes and may be decreased in chronic lead

405

toxicosis, as may the MCHC, resulting in a microcytic, hypochromic anemia.

Markers of Erythrocyte Toxicity Biomarkers of erythrocyte toxicity include quantitative and qualitative evaluation of erythrocytes for abnormalities in number and/or morphology and evaluation for presence of abnormal compounds. Packed cell volume, or hematocrit (PCV, Hct), is the value representing the percentage of erythrocytes in the blood. Increases in PCV can occur following exposure to bone marrow stimulants such as erythropoietin or with exposure to toxicants that increase erythropoietin levels (e.g., cobalt) (Simonsen et al., 2012). Decreased PCV can occur following exposure that causes damage to mature erythrocytes or that damages or inhibits replication of erythrocyte progenitors within the bone marrow. Erythrocytic toxicants may cause identifiable alteration in erythrocyte morphology, which can give clues as to the type of toxicant that caused the damage (Table 23.3). For instance, lead toxicosis is sometimes associated with the presence of relatively large numbers of nucleated red blood cells or with basophilic stippling of erythrocytes, and echinocytosis is a prominent feature in many envenomations (Flachsenberger et al., 1995). Similarly, the presence of methemoglobin and/or Heinz bodies in erythrocytes is suggestive of injury due to oxidative compounds such as nitrites, chlorates, and phenols. In addition to reduced erythrocyte numbers and morphologic alterations, oxidative erythrocyte injury resulting in hemolysis will cause elevations in total serum bilirubin secondary to hemolysis. Some toxicants cause no visible morphological change to red blood cells, but instead alter oxygencarrying capacity to such a degree as to cause lifethreatening hypoxia to the patient. Carbon monoxide binds to hemoglobin with high affinity, shifting the oxygen dissociation curve to the left, preventing oxygen delivery to tissues, and leaving no apparent morphological change in the erythrocyte, although grossly visible cherry red mucous membranes and blood are clues to the presence of hyperoxygenated blood (Guzman, 2012). Carboxyhemoglobin levels can be quickly measured in most hospital settings; levels over 20% are generally associated with signs of toxicosis, including shortness of breath, headache, and dizziness, whereas levels over 50% can be lethal. Lead poisoning is a significant concern in many parts of the world because of past and/or current use of leadbased paints and gasoline (Liu et al., 2008). Lead alters erythrocyte function through interference with heme synthesis. Inhibition of d-aminolevulinic acid dehydratase and ferrochetolase interferes with the insertion of lead into the protoporphyrin ring during heme

II. SYSTEMS TOXICITY BIOMARKERS

406 TABLE 23.2

23. BLOOD AND BONE MARROW TOXICITY BIOMARKERS

Biomarkers of Hematotoxicity (See Also Table 22.1 for Additional Leukocytes Biomarkers)

Assay

Matrix

Endpoint

Example Toxicant

Altered M:Ea

Alkylating agents ([), azathioprine ([), phenols (Y)

Cellularity Y

Busulfan

Cellularity [

Cobalt

BIOMARKERS OF TOXIC EFFECTS ON BONE MARROW Bone marrow evaluation

Bone marrow

Morphologic alterations

Methotrexate (Megaloblastosis)

Progenitor cell colony formation

Bone marrow, blood

Colony formation Y

Clopidogrel, lindane

Metabonomics profiles

Urine

Altered metabolite profiles

Benzene (biomarker of exposure)

Proteomic profiles

Serum, blood

Altered protein profiles

Benzene

Toxicogenomic assays

Blood cells, bone marrow

Altered gene expression

Benzene, cisplatin, carboplatin

Glycophorin A gene loss mutation assay

Erythrocytes

Increased

Benzene

BIOMARKERS OF TOXIC EFFECTS ON HEMOGLOBIN d-Aminolevulinic acid levels

Urine

Increased levels

Lead

d-Aminolevulinic acid dehydratase activity

Blood

Decreased activity

Lead

Mean cell hemoglobin concentration (MCHC)

Blood

Decreased levels

Lead

Coproporphyrin

Urine

Increased levels

Lead

Zinc protoporphyrin

Blood

Increased levels

Lead

Blood

Increased Hct

Cobalt

Decreased Hct

Oxidants, lindane

Mean cell volume (MCV)

Blood

Decreased MCV

Lead

Reticulocyte count

Blood

Increased

Oxidants causing hemolysis (e.g., arsine)

Carboxyhemoglobin level

Blood

Increased

Carbon monoxide

Methemoglobin level

Blood

Increased

Oxidants (e.g., methylene blue)

Heinz body preparation

Blood

Increased

Oxidants (e.g., nitrites)

BIOMARKERS OF TOXIC EFFECTS ON ERYTHROCYTES Hematocrit/Packed cell volume

BIOMARKERS OF TOXIC EFFECTS ON THROMBOCYTES Bleeding time (BT)

In vivo assay

Prolonged

Aspirin

Platelet count

Blood

Decreased

Heparin

Direct platelet assays

Blood

Decreased activity

Aspirin

Increased neutrophils

Corticosteroids

Decreased neutrophils

Phenothiazine

Increased eosinophils

L-tryptophan

Decreased eosinophils

Corticosteroids

Increased monocytes

Corticosteroids

Increased basophils

Allergens

BIOMARKERS OF TOXIC EFFECTS ON LEUKOCYTES Leukocyte count

Blood

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407

MARKERS OF HEMATOPOIETIC/HEMATOLOGIC TOXICITY

TABLE 23.2

Biomarkers of Hematotoxicity (See Also Table 22.1 for Additional Leukocytes Biomarkers)dcont’d

Assay

Matrix

Endpoint

Example Toxicant

BIOMARKERS OF TOXIC EFFECTS ON HEMOSTASIS Activated partial thromboplastin time (APTT)

Blood

Prolonged APTT

Warfarin and related anticoagulants

Coagulation factor assays

Blood

Y Factor V

Streptomycin, Penicillins

Y Factor VIII

Nitrofurazone

Y von Willebrand factor

Ciprofloxacin

Prothrombin time (PT, OSPT)

Blood

Prolonged PT

Warfarin and related anticoagulants

Proteins induced by vitamin K antagonism (PIVKA)

Blood

Increased PIVKA

Warfarin and related anticoagulants

a

Myeloid:Erythroid ratio.

TABLE 23.3

Toxicant-Induced Erythrocyte Abnormalities

Erythrocyte Abnormality

Description

Significance

Example Toxicant

Basophilic stippling

Dark blue to purple dots or specks

Represents aggregated ribosomes

Lead-induced inhibition of pyrimidine 50 -nucleotidase results in decreased RNA degradation

Eccentrocyte

Eccentric dense staining hemoglobin with adjacent clear crescent or edge

Represents fusion of membranes damaged by oxidants

Nitrites

Echinocyte

Sharp, spiny membrane projections

Represents alterations to lipid membrane

Crotalid venom

Heinz body

Pale, rounded protruding defect in membrane; dark blue with new methylene blue stain

Represents precipitated hemoglobin due to oxidative injury

Nitrites, methylene blue

Nucleated erythrocyte

Presence of dark nucleus in red blood cell; basophilic tint to cytoplasm

Represents accelerated erythropoiesis and early release from bone marrow

Lead

Reticulocyte

Aggregated or punctated basophilic staining of cytoplasm

Represents residual RNA; accelerated erythropoiesis

Oxidant-induced hemolytic anemia

Siderotic granules

Fine granular basophilic inclusions

Represents iron accumulation in damaged mitochondria

Lead

Spherocyte

Decreased central pallor, decreased diameter

Represents membrane loss

Snake envenomations

formation (Sakai, 1995). Biomarkers of lead toxicosis include elevations in urinary d-aminolevulinic acid and coproporphyrin levels, increased zinc protoporphyrin levels in the blood, decreased d-aminolevulinic acid dehydratase activity, and decreased levels of erythrocyte hemoglobin (Jangid et al., 2012).

Markers of Platelet Toxicity Toxic effects on platelets can be reflected by decreased platelet numbers and/or decreased platelet function, both of which can lead to increased incidence of

bleeding due to loss of a critical hemostatic “plug” in the face of vascular injury. Decreased platelet numbers are readily identified during routine CBC analysis, although spurious thrombocytopenia can occur due to platelet clumping within the blood sample (Stockham and Scott, 2008). Bone marrow analysis may be helpful in determining if decreased platelet numbers are due to increased consumption or decreased megakaryocyte production. Decreased platelet function, such as occurs with cyclooxygenase inhibitors such as aspirin, can be measured using direct assays of platelet function. Bleeding time is a quick and rudimentary test of platelet

II. SYSTEMS TOXICITY BIOMARKERS

408

23. BLOOD AND BONE MARROW TOXICITY BIOMARKERS

function, which is often done as a prescreening test to determine if further platelet and/or coagulation assays are indicated. A small cut is made in the skin with a lancet or needle and the amount of time that it takes for the bleeding to cease is measured.

MARKERS OF BONE MARROW TOXICITY Bone Marrow Evaluation Evaluation of bone marrow can be done using histopathology, cytology, or flow cytometry. Histopathological evaluation of hematoxylin and eosin-stained (H&E) sections of bone marrow tissue may be used as an initial screening tool but has some limitations, so it is prudent to prepare bone marrow smears at the time of autopsy or biopsy for cytological examination in case further evaluation is needed (Elmore, 2006). Histopathology can provide estimates of cellular density, myeloid/erythroid (M:E) ratios, amount of hemosiderin present, and abnormalities in the numbers of megakaryocytes, adipocytes, and bone marrow stromal cells. Additionally, abnormalities such as necrosis, hemorrhage, fibrosis, granulomas, neoplasia, or alterations in endosteum, bone, and vasculature can be detected (Reagan et al., 2011). Because many of the mononuclear cells in bone marrow “all look alike” under H&E, differentiating the lymphoid lineage cells from early myeloid precursors can be difficult. Cytological examination of Romanowsky-stained bone marrow smears may be more rewarding in cases where there is a need to differentiate early hematopoietic precursors, to correlate changes in peripheral cell numbers with bone marrow hyper- or hypocellularity, to investigate suspected abnormal erythropoiesis, to differentiate lymphoid from erythroid precursors, or to determine maturation indices of one or more cell lines (Elmore, 2006). Flow cytometry may be used in addition to or instead of cytology to further characterize TABLE 23.4

alterations in bone marrow cells. However, although flow cytometry can readily classify cells into the major cell lineages and produce rapid counts of large numbers of cells, it does not evaluate cellular morphology; for this reason, flow cytometric methods are generally used in addition to either cytology or histopathology (Reagan et al., 2011). Bone marrow evaluation generally starts with estimation of the relative cellularity and the M:E ratio, which compares the relative proportions of myeloid cells to erythrocytic cells (Table 23.4). Generalized hypocellularity of bone marrow can occur due to infection, irradiation, or exposure to various drugs or toxicants such as benzene, cephalosporins, chloramphenicol, or phenylbutazone (Bloom and Brandt, 2008; Bloom et al., 1987). Because immature hematopoietic cells are difficult to differentiate, the M:E ratio obtained by visual inspection is a relatively subjective estimation (Stockham and Scott, 2008). In most healthy mammals, the M:E ratio trends toward slightly >1, i.e., a somewhat greater proportion of myeloid cells (Elmore, 2006). Increases in M:E ratio can be due to increases in myeloid cells or decreases in erythrocytic cells, which can be determined by estimating the overall cellularity of the marrow. Hypercellular marrow with increased M:E ratio suggests myeloid hyperplasia, whereas hypocellularity of hematopoietic cells and increased M:E ratio suggests erythroid hypoplasia. The most common cause of myeloid hyperplasia is increased demand during times of acute or chronic inflammation, which oftentimes will also be reflected in the results of a CBC. Erythroid hypoplasia can occur in response to drugs such as isoniazid or azathioprine (Thompson and Gales, 1996). Decreases in M:E ratio can occur during times of erythroid hyperplasia, such as occurs in response to acute hemorrhagic anemias or cobalt exposure, or to myeloid hypoplasia, such as may occur with carbamazepine or clozapine (Simonsen et al., 2012; Bloom and Brandt, 2008).

Bone Marrow Evaluation, Assessments, and Potential Causative Agents

M:E Ratio

Cellularity

Assessment

Example Causes

Decreased

Hypercellular

Erythroid hyperplasia

Erythropoietin therapy or excess Hemorrhagic anemia Hemolytic anemia: e.g., acetanilide, nitrofurantoin, phenol, sulfonamides Cobalt

Decreased

Hypocellular

Granulocytic hypoplasia

Alkylating agents, benzene, carbamazepine, cisplatin, clozapine, isoniazid, lindane, methimazole, nitrosourea

Normal

Hypocellular

Bone marrow hypoplasia

Alkylating agents, benzene, chloramphenicol, chlorinated hydrocarbons, diclofenac, mycotoxins, sulfonamide

Increased

Hypercellular

Granulocytic hyperplasia

Inflammation leukomogenic response: e.g., alkylating agents, azathioprine, benzene (chronic), bleomycin, high-dose ionizing radiation, procarbazine

Increased

Hypocellular

Erythroid hypoplasia

Chronic renal disease (decreased erythropoietin) azathioprine, estrogen, isoniazid, phenytoin

II. SYSTEMS TOXICITY BIOMARKERS

MARKERS OF HEMOSTATIC TOXICITY

Bone marrow examination should include evaluation of all of the major cellular components for anomalies in morphology. Toxicants such as ethanol, lead, or isoniazid can cause defects in synthesis of the porphyrin ring, resulting in failure of iron to incorporate into heme and leading to precipitation of iron within erythrocyte mitochondria, resulting in sideroblastic anemia (Bloom and Brandt, 2008). Megaloblastic anemia develops when folate or vitamin B12 deficiencies occur either due to nutritional deficiency or due to effects of toxicants such as phenytoin, phenobarbital, and sulfasalazine. In megaloblastic anemia, asynchronous development of nucleus and cytoplasm in erythroid precursors results in cells with abundant cytoplasm and immature, enlarged nuclei displaying exaggerated chromatin patterns (Stockham and Scott, 2008).

Progenitor Cell Colony Formation Evaluation of hematopoietic cells in early differentiation stages is difficult by means of light microscopy and flow cytometry due to lack of distinctive cell markers. Bone marrow and blood cells cultured in a semisolid methylcelluloseebased medium containing appropriate growth factors will proliferate and differentiate into colonies of maturing hematopoietic cells (Wognum et al., 2013; Masenini et al., 2012). Classification and enumeration of the colonies is performed by light microscopy or flow cytometry and allows for the quantification of erythroid, myeloid, lymphoid, and megakaryocytic cell lineages to detect toxicant-induced increases or decreases in specific hematopoietic cell lines.

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such as warfarin and related compounds such as diphacinone and brodifacoum. These anticoagulants inhibit vitamin K epoxide reductase, a vital enzyme in the regeneration of vitamin K, resulting in an inability to generate vitamin K-dependent coagulation factors II, VII, IX, and X (Stafford, 2005). As these coagulation factors are depleted, coagulation becomes impaired and incomplete coagulation proteins are released into the circulation. Defects in coagulation can be measured using several assays, including activated partial thromboplastin time (APTT), prothrombin time (PT), proteins induced by vitamin K antagonism or absence (PIVKA), and measurement of individual coagulation factors. The APTT, PT, and PIVKA assays are the most frequently used for detecting early anticoagulantinduced coagulopathy (Stockham and Scott, 2008). The APTT and PT assays are performed by incubating test plasma with partial thromboplastin (APTT) or thromboplastin (PT) along with other intermediates and measuring the time until clot formation occurs. The PIVKA assay used in human diagnostic laboratories directly measures the incompletely carboxylated coagulation factors, whereas the PIVKA assay used in veterinary medicine (Owren’s thrombotest) is essentially a modified PT assay (Stockham and Scott, 2008). Individual coagulation factors can be measured, and although these assays are more commonly used for suspected inherited coagulation factor deficiencies (e.g., hemophilia), certain xenobiotics can result in deficiencies of individual coagulation factors; for instance, ciprofloxacin

MARKERS OF HEMOSTATIC TOXICITY Prevention of excessive loss of blood is essential to maintain the integrity of the hematologic system; an elaborate and overlapping coagulation system, involving platelets and clotting factors, has evolved to ensure prompt repair of vascular leakage. Hemostasis involves sequential activation of serine proteases, culminating in the formation of fibrin clots to seal defects in the vasculature (Fig. 23.2). Although hypercoagulable states can occur secondary to exposure to some chemotherapeutic agents (e.g., tamoxifen), most alterations in hemostatic ability are due to deficiencies in one or more components of the coagulation cascade resulting in ineffective coagulation and predisposing to uncontrolled hemorrhage. Decreases in coagulation factors can occur due to reduced synthesis or increased clearance. Perhaps the most common cause of decreased coagulation factor synthesis is due to vitamin K deficiency induced by anticoagulants

FIGURE 23.2 Schematic diagram of the coagulation cascade. Coagulation initiates with activation of intrinsic factor XII to XIIa and/ or activation of the combined factors VII and III to VIIaIIIa. XIIa initiates a series of factor activations culminating in production of activated factor VIII. Factors VIIaIIIa and/or VIIa then activate factor X, triggering activation of factor V, which triggers the conversion of prothrombin to thrombin. Thrombin acts on fibrinogen to form fibrin. Enzymatic action by activated factor XIII in presence of ionized calcium results in cross-linkage of fibrin strands. *indicates vitamin Kdependent factors.

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23. BLOOD AND BONE MARROW TOXICITY BIOMARKERS

has been associated with transient acquired von Willebrand syndrome due to increased proteolysis of von Willebrand factor (Michiels et al., 2011).

CONCLUDING REMARKS AND FUTURE DIRECTIONS Our understanding of the intricate connections responsible for the normal functioning of the blood and bone marrow has expanded tremendously over the last few decades. With this expanding knowledge has come the realization that this delicately balanced system is highly susceptible to injury induced by xenobiotics such as pharmaceuticals and environmental toxicants. Toxicant-induced bone marrow injury can have serious repercussions throughout the body and can put the host at risk of infection, hypoxic injury, or uncontrolled hemorrhage. This ripple effect underscores the need to determine that new and existing pharmaceuticals and environmental compounds pose minimal risk to blood or bone marrow components. Future research to further our knowledge of the mechanisms of toxicity of hematopoietic tissues will enable the development of pharmaceuticals, pesticides, and other compounds that pose less of a risk to humans, other animals, and the environment. Newer technologies in research such as toxicogenomics, proteomics, and metabonomics will allow further progress in mechanistic investigation and risk assessment. Toxicogenomic profiles have been proposed as potentially useful biomarkers for exposure to various toxicants. For instance, circulating reticulocyte expression of the hemoglobin beta chain complex, aminolevulinic acid synthase 2, and cell division cycle 25 homolog B genes was altered following exposure of rats to myelosuppressive agents such as linezolid, cisplatin, and carboplatin, suggesting that this pattern of gene expression may pose a useful biomarker for myelosuppressive anemia in rats (Uehara et al., 2011). Microarray evaluation of the hepatic expression of six genes (Alas2, beta-glo, Eraf, Hmox1, Lgals3, and Rhced) showed high negative correlation with red blood cell counts and high positive correlation with total serum bilirubin levels in rats with drug-induced hemolysis, suggesting that these genes may be useful biomarkers for hemolytic anemia (Rokushima et al., 2007). Further validation of these and other toxicogenomic profiles will be required to determine if similar changes in gene expression are shared among other mammalian species. Benzene is a potent myelotoxic compound associated with aplastic anemia, acute myeloid leukemia, and other blood disorders in humans (Zhang et al., 2010). In humans, increased levels of gene duplication mutations in glycophorin A have been proposed as a potential biomarker

for cumulative exposure to benzene, although sensitivity issues have been raised (Rothman et al., 1995; Smith and Rothman, 2000). Proteomics is the branch of toxicogenomics that studies alterations in protein levels and posttranslational protein modifications that result from altered gene expression caused by exposure to toxicants (Joo et al., 2003). Two proteins, platelet factor 4 and connective tissue activating peptide III, have been found to be consistently downregulated in patients exposed to benzene when compared with control individuals and are being investigated as potential biomarkers for the early biologic effects of benzene (Zhang et al., 2010). Similarly, downregulation of platelet basic protein and apolipoprotein B100 has been detected in humans with hematotoxicity due to exposure to benzene (Huang et al., 2012). Plasma haptoglobin levels in the plasma of pancreatic cancer patients have been shown to be correlated to risk of hematologic adverse events from the chemotherapeutic agent gemcitabine (Matsubara et al., 2009) Further work is needed to determine if these potential biomarkers will prove to be reliable in determining risks of benzene exposure. Metabonomics is a means of metabolic profiling to determine biological markers for mechanistic and diagnostic study in toxicology, pharmacology, and biomedicine. Benzene and its metabolites have been found in breath, blood, and urine, and levels in these matrices have been used as biomarkers for exposure and risk assessment in humans (Weisel, 2010). In mice, metabonomic profiles of benzene metabolites in urine have been further utilized as a sensitive tool to detect benzene-induced toxicity (Sun et al., 2012).

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