Toxicity Study of DC-D3T in Rats

Toxicity Study of DC-D3T in Rats

PRE-CLINICAL TOXICOLOGICAL EVALUATION OF 5,6-DIHYDRO-4H-CYCLOPENTA-1,2-DITHIOLE-3-THIONE, A POTENTIAL CANCER CHEMOPREVENTIVE DRUG, IN RATS
Rajabather Krishnaraj1, Izet M. Kapetanovic2, Robert L. Morrissey3, Thomas Kensler4, James Crowell2, Barry S. Levine1
1Toxicology Research Laboratory, University of Illinois at Chicago, Chicago, IL; 2NCI, Bethesda, MD; 3Pathology Associates, Chicago, IL; 4Johns Hopkins University, Baltimore, MD.

ABSTRACT

The purpose of this study was to examine the in vivo toxicity of 5,6-dihydro-4H-cyclopenta-1,2-dithiole-3-thione (DC-D3T), a potential cancer chemopreventive drug that may act primarily as a phase II enzyme inducer, in normal CD rats. Based on a 14-day range finding study, DC-D3T was administered daily by oral gavage (20/group/sex) for 28 days at 0, 3,12 and 50 mg/kg bwt/day. Endpoints included daily clinical signs, weekly body weight and food consumption, week 4 clinical chemistry, hematology, coagulation, ophthalmology, and necropsy at week 5 and histopathology. The mean plasma DC-D3T levels (measured 2 hours post-dosing in week 4) in the 3 (low), 12 (mid) and 50 (high) mg/kg/day dose groups were 0.28, 0.77 and 1.83 μg/mL for males and 0.24, 0.70 and 2.05 μg/mL for females, respectively. No mortalities or clinical signs of toxicity were seen. Reduced body weight gains and food consumption occurred in the high dose group males. At the high dose, hepatic effects were seen: elevated serum ALT, bilirubin (in the high and mid dose group females), protein, albumin, globulin, cholesterol and decreased triglycerides, and hepatomegaly. Mild renal injury was inferred from hypochloremia in the high dose group and creatininemia in high and mid dose group males. Decreased weights of heart and thyroid/parathyroids noted in the high dose group rats were unaccompanied by histologic changes. Splenomegaly (high dose), dose-dependent increases in the incidence and severity of follicular center hyperplasia of spleen and mesenteric lymph node (at low, mid and high doses), mild monocytosis and reticulocytosis (high dose group females) were also observed. Chronic perivascular inflammation (presence of lymphocytes around small arteries) of epididymis and kidney was noted at 3, 12 and 50 mg/kg/day of DC-D3T. The preliminary data on the effect of DC-D3T on lymphoid tissues could be interpreted as either general immunomodulation or toxicity of DC-D3T. Additional studies are needed to define the threshold for the tissue inflammatory reactions and possible direct immunologic effects of DC-D3T. Under the conditions of this study, a NOEL was not found for DC-D3T in male and female rats. (Supported by NCI contract No.N01-CN-0512).

CONCLUSIONS

* Toxicity of 5,6-dihydro-4H-cyclopenta[1,2]-dithiole-3-thione (DC-D3T) was tested in CD® rats after 28 days of daily gavage administration at 0, 3 (low dose), 12 (mid dose) and 50 (high dose) mg/kg/day.
* Neither mortalities nor drug-related clinical signs were seen at any dose.

* Reductions in weekly body weight gains accompanied by reduced food consumption occurred in the high dose group males, culminating in 25% decrease in total body weight gain.
* Elevated serum total bilirubin levels (high & mid dose group females) and elevated %reticulocytes (high & mid dose group males) were noted. There was no unequivocal evidence for mild hemolytic anemia.
* Mild hepatic injury was evident from the raised serum ALT levels (high dose group), hepatomegaly (high & mid dose group), and a dose-dependent increase in the incidence & severity of centrilobular hypertrophy in males. Hyperproteinemia, hypercholesterolemia and hypotriglyceridemia were also noted at high dose.
* Mild renal injury was suggested by elevated serum creatinine (at high dose), and chloride levels (in high and mid dose group males).
* Mean plasma drug levels in the low, mid and high dose groups were 0.284, 0.767 and 1.831 µg/mL (males) and 0.239, 0.700 and 2.045 µg/mL (females).
* Hyperglobulinemia, splenomegaly (high dose group), dose-dependent increase in the incidence and severity of follicular center hyperplasia of spleen (all DC-D3T treated groups) and mesenteric lymph node (low, mid and high dose group females) were also noted. These effects were accompanied by dose-dependent chronic perivascular inflammation of epididymis and kidney in all dose groups. These findings could be interpreted as either immunostimulatory or toxic effect of DC-D3T. A comprehensive evaluation of the immunomodulatory effect of this chemopreventive agent is needed.

* Under the conditions of this study, a No-Observed-Effect-Level was not found for DC-D3T.

Summary of Treatment-Related Lesions
* Incidence (severity range)
LYMPH NODE, MESENTERIC

-Hyperplasia, follicular center
-Inflammation, chronic, perivascular
-Hyperplasia, follicular center
-Hypertrophy, centrilobular
-Vacuolation, centrilobular

ORGAN - lesion
Dose (mg/kg/day DC-D3T)
Summary Of DC-D3T-Treatment Related Changes
Hematology
Clinical Chemistry
Food Consumption
Total Body Weight Gain
EXPERIMENTAL DESIGN

During the quarantine/pretest period, 80 male and 80 female rats were randomized using a computer-generated randomization program, stratified on the basis of body weight, and animals were assigned to 4 treatment groups of 20 males and 20 females per group. The study was stagger-started over two consecutive days beginning with the males.
Dose Concentration
Dose Volume
LIST OF ORGANS AND TISSUES EVALUATED FOR LESIONS

Abnormal lymph nodes Pancreas
*Adrenal glands Pituitary
Aorta (thoracic) Prostate
*Brain (fore-, mid-, hind-) Salivary gland (mandibular)
Cecum Sciatic nerve
Colon Seminal vesicles
Duodenum Skeletal muscle
Epididymides Skin
Esophagus Spinal cord (cervical and Eyes thoracolumbar segments)
Femur (with marrow) *Spleen
Gross lesions Sternum (with marrow)
*Heart Stomach
Ileum *Testes
Jejunum Thymus
*Kidneys *Thyroid/parathyroids
*Liver *Tissue masses
*Lungs/Bronchi Trachea
Lymph node (mesenteric) Urinary bladder
Mammary gland Uterus (corpus and cervix)
*Ovaries/Fallopian tubes Vagina
* Organs weighed.

Toxicity Study of DC-D3T in Rats.ppt

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Basic Clinical Chemistry Lab Testing

Clinical Laboratory Testing – Basic Clinical Chemistry
Presentation lecture by:Cecile Sanders, M.Ed., MT(ASCP),
CLS (NCA)

Purpose of Clinical Chemistry Tests

*Measure levels of substances found normally in human blood that have biological functions. Examples: Glucose, Calcium
*Detect or measure non-functional metabolites or waste products. Examples: Creatinine, Blood Urea Nitrogen (BUN)

Clinical Laboratory Testing - Basic Clinical Chemistry

*Detect or measure substances that indicate cell damage or disease. Examples: Liver enzymes, such as ALT, Cardiac enzymes, such as CK-MB
*Detect or measure drugs or toxic substances. Examples: Dilantin, Drugs of abuse screen
* Types of Specimens for Chemical Analysis
*Whole blood, serum or plasma. The most common specimen is serum, collected in a tube with no anticoagulant so that the blood will clot.
*Urine – often 24 hour collections
*Others – Cerebrospinal Spinal Fluid (CSF) and other fluids

* Collection and Handling of Blood Specimens for Chemical Analysis
*Blood collection tubes for obtaining serum - Serum Separator Tubes (SST) do not have an anticoagulant but do contain a gel substance which will form an interface between the clot and the serum when the blood specimen is centrifuged. These tubes are sometimes referred to as “Tiger Tops”.

*Blood collection tubes for obtaining plasma
*Patient preparation; time of collection; & effects of eating on chemistry analysis

*Some specimens are increased or decreased after eating (ex. Glucose, triglycerides), so it is important to know what the test and collection method call for. Specimens for these tests are usually collected in a fasting state.

*Sometimes serum or plasma appears lipemia (milky) after a patient has eaten a fatty meal. Lipemia affects most chemistry analyses. The blood must be recollected when the patient is fasting.


* Clinical Chemistry Tests
*Normal or Reference Values – range of values for a particular chemistry test from healthy individuals
*Chemistry Panel grouping – some tests are “bundled” according to the system or organ targeted. Examples: thyroid panel, liver panel, cardiac panel, kidney panel, basic metabolic panel, etc.

* Commonly Performed Chemistry Tests or Analytes
*Proteins – essential components of cells and body fluids. Some made by body, others acquired from diet. Provides information about state of hydration, nutrition and liver function, since most serum proteins are made in the liver.

*Electrolytes – sometimes called “lytes”
*Includes sodium (Na), potassium (K), chloride (Cl) and bicarbonate (HCO3-)
*Collectively these have a great effect on hydration, acid-base balance and osmotic pressure as well as pH and heart and muscle contraction
*Levels differ depending on if inside vs. outside cells
*Important in transport of substances into and out of cells

*Minerals
*Calcium
*Used in coagulation and muscle contraction
*99% is in skeleton and is not metabolically active
*Influenced by vitamin D, parathyroid hormone, estrogen and calcitonin
*Hypercalcemia – occurs in parathyroidism, bone malignancies, hormone disorders, excessive vitamin D, and acidosis; may cause kidney stones
*Hypocalcemia – can cause tetany; occurs in hypoparathyroidism, vitamin D deficiency, poor dietary absorption and kidney disease

*Phosphorus
*80% in bone and rest in energy compounds such as ATP
*Influenced by calcium and certain hormones
*Iron
*Essential for hemoglobin
*Deficiency results in anemia; may be caused by lack of iron in diet, poor absorption, poor release of stored iron or loss due to bleeding
*Increased in hemolytic anemia, increased iron intake or blocked synthesis of iron-containing compounds, such as in lead poisoning


*Kidney Function Tests
*Serum Creatinine
*Best test for overall kidney function; not affected by diet or hormone levels
*Waste product of muscle metabolism
*Serum creatinine rises when kidney function is impaired

*BUN (Blood Urea Nitrogen)
*BUN is surplus amino acids that are converted to urea and excreted by kidneys as a waste product
*BUN influenced by diet and hormones, so it is NOT as good an indicator of renal function as serum creatinine levels
*BUN increased in kidney disease, high protein diet, and after administration of steroids
*BUN decreased in starvation, pregnancy and in persons on a low protein diet

*Uric Acid
*Formed from breakdown of nucleic acids and excreted as a waste product by kidneys
*Increased in kidney disease, but most often used to diagnosis gout (pain in joints, mainly big toe, due to precipitated uric acid crystals)
*Also increased in increased cell destruction, such as after massive radiation or chemotherapy

*Liver Function Tests
*Liver functions:
*Synthesizes glycogen from glucose
*Makes plasma proteins (albumin, lipoproteins, coagulation proteins)
*Forms cholesterol and degrades it into bile acids, which emulsifies fats for absorption
*Stores iron, glycogen, vitamins and other substances
*Destroys old blood cells and recycles components of hemoglobin

*Total Bilirubin
*Waste production of hemoglobin breakdown
*Increased in excessive RBC breakdown, such as hemolytic anemia, or impaired liver function or some sort of obstruction, such as a tumor or gall stone

*Liver Enzymes – levels increase following damage to liver tissues
*Alkaline Phosphatase (ALP or AP) - Greatly increased in liver tumors and lesions; moderately increased in diseases such as hepatitis
*Alanine Aminotransferase (ALT; formerly called SGPT) - Increases up to 10x in cirrhosis, infections or tumors and up to 100x in viral or toxic hepatitis


*Asparate Aminotransferase (AST; formerly called SGOT) - Increased in liver disease, but also in heart attacks
*Gamma Glutamyl Transferase (GGT) - Often used to monitor patients recovering from hepatitis and cirrhosis
*Lactate Dehydrogenase (LD) - Increased in liver disease and following heart attacks

*Cardiac Function Tests
*Creatine Kinase (CK) - Widely used to diagnosis and monitor heart attacks
*Troponins
* Only present in heart muscle, making it a more accurate indicator of heart attack than CK
* Cardiac Troponin T (cTnT)
* Cardiac Troponin I (cTnI)
*Lipid Metabolism Tests
*Cholesterol
* Present in all tissues
* Serves as the skeleton for many hormones
* Recommended to be less than 200 mg/dL in adults)
* LDL = “bad” cholesterol; HDL = “good” cholesterol

*Triglycerides
* Main storage form of lipids, comprising 95% of fat tissue
* Hyperlipidemia – having high blood levels of triglycerides – may increase risk of heart attack
*Carbohydrate Metabolism Tests
*Glucose - Largely regulated by insulin

*Thyroid Function Tests

*Thyroid Stimulating Hormone (TSH) - Inverse relationship to thyroid function (the higher the TSH, the lower the thyroid function and vice versa)
*Other less common thyroid tests include T3 and T4
*Hypothyroidism – underactive thyroid gland
*Hyperthyroidism – overactive thyroid gland

Basic Clinical Chemistry.ppt

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Blood - Overview, Composition

Blood
Presentation lecture by:Melody Holmes, MT,
Collin County Community College District

Overview of Blood Circulation

Composition of Blood

* Tissue type
* Composition
* Formed elements
o Erythrocytes
o Leukocytes
o Platelets
* Hematocrit

Components of Whole Blood
Physical Characteristics and Volume
* Characteristics
* Color
* pH
* Temperature
* Percentage of body weight
* Average volume

Functions of Blood
* Distribution
* Regulation
* Protection

Distribution
* Transportation
o Gases
o Nutrients
o Metabolic wastes
o Hormones
* System Maintenance
o Body temperature
o pH
o Fluid volume

Protection
* Blood loss
o Plasma proteins and platelets
o Clot formation
* Infection
o Antibodies
o Complement proteins
o WBCs

Blood Plasma
* Solutes
o Proteins
+ Albumin, globulins, clotting proteins
o Lactic acid, urea, creatinine
o Organic nutrients
+ Glucose, carbohydrates, amino acids
o Electrolytes
+ Sodium, potassium, calcium, chloride, bicarbonate
o Respiratory gases
+ Oxygen and carbon dioxide
Formed Elements

* Erythrocytes, leukocytes, and platelets
o WBCs
o RBCs
* Life span
* Production

Erythrocytes

* Physical characteristics
o Shape
o Nucleation
o Organelles
* Hemaglobin
o Function
* Spectrin
o Function

Erythrocytes Gas Transport
* Structural characteristics
o Surface area
o Hemoglobin
o ATP generation
Erythrocyte Function
* RBCs are dedicated to respiratory gas transport
* Hemaglobin (Hb)
o Affinity for oxygen
o Composition
o Heme group
o Capacity

Structure of Hemoglobin
Hemoglobin (Hb)
* Oxyhemoglobin
* Deoxyhemoglobin
* Carbaminohemoglobin

Production of Erythrocytes
* Hematopoiesis
* Red bone marrow
o Axial skeleton and girdles
o Epiphyses of the humerus and femur
* Hemocytoblasts

Production of Erythrocytes: Erythropoiesis
* Hemocytoblast → Proerythroblast
* Proerythroblasts → Early Erythroblasts
* Developmental pathway
o 1 – ribosome synthesis in early erythroblasts
o 2 – Hb accumulation in late erythroblasts and normoblasts
o 3 – ejection of the nucleus from normoblasts and formation of reticulocytes
* Reticulocytes → Mature Erythrocytes

Erythropoiesis: Regulation and Requirements
* Circulating erythrocyte number
o Too few RBCs
o Too many RBCs
* Erythropoiesis control
o Iron, amino acids, and B vitamins

Erythropoiesis: Hormonal Control
* Erythropoietin (EPO)
o Triggers
+ Hypoxia
+ Decreased oxygen availability
+ Tissue demand for oxygen
* Enhanced erythropoiesis
o Outcome
+ RBC count
+ Oxygen carrying ability
Homeostasis: Normal blood oxygen levels
Erythropoietin Mechanism
Homeostasis: Normal blood oxygen levels
Erythropoiesis: Dietary Requirements
Erythrocytes: Fate and Destruction
* Life span
* Degenerate
* Dying RBCs
o Macrophages
* Heme and globin
* Iron
Erythrocytes: Fate and Destruction
* Heme → Bilirubin
* Bilirubin → Bile
* Bile → Urobilinogen
* Stercobilin
* Globin
* Hb
Erythrocyte Disorders
Anemia: Insufficient Erythrocytes
* Hemorrhagic anemia
* Hemolytic anemia
* Aplastic anemia
* Iron-deficiency anemia
o Secondary result
o Iron-containing foods
o Iron absorption
* Pernicious anemia
o Vitamin B12
o Intrinsic factor
* Treatment
o B12
o Nascobal
Anemia: Decreased Hemoglobin Content
Anemia: Abnormal Hemoglobin
* Sickle-cell anemia
o HbS
+ Amino acid substitution
o Result

Polycythemia

* Polycythemia
* Three main polycythemias
o Polycythemia vera
o Secondary polycythemia
o Blood doping

Blood.ppt

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