Germline genome protection: implications for gamete quality and germ cell tumorigenesis.

BACKGROUND: Germ cells have a unique and critical role as the conduit for hereditary information and therefore employ multiple strategies to protect genomic integrity and avoid mutations. Unlike somatic cells, which often respond to DNA damage by arresting the cell cycle and conducting DNA repair, germ cells as well as long-lived pluripotent stem cells typically avoid the use of error-prone repair mechanisms and favor apoptosis, reducing the risk of genetic alterations. Testicular germ cell tumors, the most common cancers of young men, arise from pre-natal germ cells. OBJECTIVES: To summarize the current understanding of DNA damage response mechanisms in pre-meiotic germ cells and to discuss how they impact both the origins of testicular germ cell tumors and their remarkable responsiveness to genotoxic chemotherapy. MATERIALS AND METHODS: We conducted a review of literature gathered from PubMed regarding the DNA damage response properties of testicular germ cell tumors and the germ cells from which they arise, as well as the influence of these mechanisms on therapeutic responses by testicular germ cell tumors. RESULTS AND DISCUSSION: This review provides a comprehensive evaluation of how the developmental origins of male germ cells and their inherent germ cell-like DNA damage response directly impact the development and therapeutic sensitivity of testicular germ cell tumors. CONCLUSIONS: The DNA damage response of germ cells directly impacts the development and therapeutic sensitivity of testicular germ cell tumors. Recent advances in the study of primordial germ cells, post-natal mitotically dividing germ cells, and pluripotent stem cells will allow for new investigations into the initiation, progression, and treatment of testicular germ cell tumors.

Principles of hematotoxicology: laboratory assessment and interpretation of data.

The toxicologic evaluation of the hematopoietic system is part of most preclinical and clinical safety studies and has become routine in monitoring a variety of novel and conventional therapies in humans and animals. As with spontaneous disease, iatrogenic blood dyscrasias may be primary but are frequently secondary to other tissue toxicity. The latter tendency makes this easily accessible tissue particularly useful in monitoring for systemic toxicity, while primary hematotoxicity ranks with liver and kidney effects as important and often limiting complications. Although the principles driving the diagnostic approach to spontaneous (clinical) blood disorders generally apply to preclinical and clinical safety studies, there are important differences, particularly regarding control of variables, feasibility of testing, and interpretation of resulting data. The luxury of studying a homogenous population of subjects free of complicating disease under controlled (uniform) laboratory and environmental conditions allows changes to be defined with greater precision and sensitivity. There are generally more options regarding the assays available and frequency of monitoring. Moreover, the hierarchy of tests applied are influenced by regulatory as well as scientific or problem-driven indications. Finally, interpretation of laboratory findings is usually based on the use of subjects as their own controls (pretreatment and sequential monitoring), comparison to a control population and well-defined reference ranges specific for the population under study, and in accordance with the principles of pathology and internal medicine.

Hepatocellular carcinoma associated with erythrocytosis and hypoglycemia in a yearling filly.

A yearling Arabian-type filly with a history of poor growth, erythrocytosis, hypoglycemia, and high liver enzyme activities was admitted to the hospital for evaluation. Three days after admission, the filly collapsed, deteriorated rapidly despite treatment, and was euthanatized. A metastatic hepatocellular carcinoma with capsular rupture and hemoperitoneum were found at necropsy. Primary liver tumors are rare in horses, and hepatocellular carcinoma has been reported in only 1 other horse. The systemic manifestations of the tumor in this filly included weakness, weight loss, inappetence, erythrocytosis with tumor production of erythropoietin, persistent hypoglycemia with normal serum insulin concentrations, serum alpha-fetoprotein (normally present only during fetal life), and terminal massive hemoperitoneum, all features of the syndrome in man.

Use of danazol for treatment of corticosteroid-resistant immune-mediated thrombocytopenia in a dog.

A routine health screen of a 7 1/2-year-old female Beagle participating as a control in a long-term study revealed thrombocytopenia. Immune thrombocytopenia was diagnosed on the basis of regenerative bone marrow findings (high number of megakaryocytes) and evidence of antiplatelet antibodies. Treatment with prednisone at dosages up to 1 mg/kg of body weight every 12 hours resulted in limited improvement, with relapses of severe thrombocytopenia thwarting attempts to taper the corticosteroid, and was further complicated by side effects of the drug. Addition of danazol to the treatment regimen (5 mg/kg, q 12 h) resulted in remission of the thrombocytopenia within 2 weeks and permitted the dosage of prednisone to gradually be reduced and discontinued. Associated with this response was a decrease in platelet-associated IgG to values comparable with control samples.

The effect of human recombinant tissue-type plasminogen activator on clinical and laboratory parameters of hemostasis and systemic plasminogen activation in the dog and the rat.

The effect of human recombinant tissue-type plasminogen activator (rt-PA) on parameters of hemostasis and systemic plasminogen activation was studied in the dog and rat. Effects on screening coagulation times, fibrinogen concentration, fibrin/fibrinogen degradation products, and plasminogen and alpha 2-antiplasmin (alpha 2-AP) activities in plasma were examined following single bolus injections of 0.5-5.0 mg/kg, single and repeated 3 hr infusions of 0.75-7.5 mg/kg and 24 hr infusions of 6.0 and 30.0 mg/kg administered intravenously to dogs. Rats receiving single or 14 daily injections of 5.0-30.0 mg/kg i.v. were similarly monitored. Systemic fibrinogenolysis (greater than 50% decrease in fibrinogen, plasminogen or alpha 2-AP values) was observed in dogs receiving greater than or equal to 1.0 mg/kg as a bolus, greater than or equal to 3.75 mg/kg (20.8 micrograms or 1.19 x 10(4) IU kg-1min-1) as a 3 hr infusion and greater than or equal to 6 mg/kg (4.2 micrograms or 2.42 x 10(3) IU kg-1min-1) as a 24 hr infusion; and in rats treated with bolus injections of 30 mg/kg rt-PA. Clinical and laboratory indications of impaired hemostasis and bleeding (anemia, prolonged coagulation times and post-mortem evidence of hemorrhage) were associated with these effects, which together were dose-dependent and influenced by the rate of infusion. The incidence of major hemorrhage was variable and limited to animals receiving prolonged (24 hr) or repeated infusions.

Cephalosporin-induced immune cytopenia in the dog: demonstration of erythrocyte-, neutrophil-, and platelet-associated IgG following treatment with cefazedone.

Cephalosporin treatment in man has been associated with a low incidence of hemolytic anemia, thrombocytopenia, and neutropenia; some cases have been shown to be immune-mediated. This triad of blood dyscrasias was also demonstrated in our laboratory in a series of toxicity studies in dogs of two cephalosporin compounds, cefonicid and cefazedone; these studies provided evidence for drug-associated immune hemolytic anemia, based on conventional laboratory tests. To further investigate possible immune mechanisms of the cephalosporin-induced cytopenias, we measured erythrocyte-associated, platelet-associated (PAIgG), and serum antineutrophil IgG over the course of cephalosporin treatment, using highly sensitive 125I-staphylococcal protein A (SPA) assays, as well as the direct antiglobulin test; we compared these findings with the hematologic changes. Intravenous treatment with high doses of cefazedone (540 mg/kg/day, increased to a maximum of 840 mg/kg/day for 4 months or until hematologic effects were evident) resulted in a high incidence of anemia (7/14), thrombocytopenia (11/14), and neutropenia (7/14). Of the affected dogs examined, 6/7 with anemia, 9/9 with thrombocytopenia, and 7/7 with neutropenia showed increased levels of the respective cell-associated antibody, compared with untreated controls. Unaffected dosed animals generally did not show these changes. In 3/3 dogs examined following remission of thrombocytopenia, PAIgG returned to levels comparable with controls; as one of these dogs suffered a relapse, increased PAIgG was again observed. Animals sacrificed during cytopenic episodes showed cytologic and histologic evidence of increased hemophagocytosis. We conclude that antibody-mediated blood cell destruction contributes to all three cephalosporin-induced cytopenias in the dog.

The effect of longterm treatment with auranofin and gold sodium thiomalate on immune function in the dog.

Although gold compounds are recognized as effective immunomodulatory agents in the treatment of rheumatoid arthritis (RA), their mechanism of action is controversial. We examined the effect of longterm treatment with 0.6-3.6 mg/kg auranofin (AF) per os q24h, or intramuscular injections of 0.5-2.0 mg/kg gold sodium thiomalate (GSTM) q3d, on 13 variables of immune function in normal dogs. None of the changes in these variables previously attributed to treatment with AF or GSTM could be demonstrated after 6-7 years' dosing. As gold compounds are effective in treating spontaneous RA in dogs, these proposed actions may not be responsible for the remittive effects of chrysotherapy in this disease.

Techniques for assessing canine mononuclear phagocyte function as part of an immunotoxicologic evaluation.

Although the dog is used extensively as a model for toxicologic investigation, few practical techniques for assessing immunotoxicity in this species have been reported. We have adapted techniques for assessing peripheral blood monocyte (PBM) function in the dog. Methods for isolating PBM were compared; a technique employing hypertonic conditioning followed by density gradient centrifugation, which resulted in greater than 90% purity and a greater yield of PBM than that obtained with other previously reported methods is described, along with methods for measuring antibody-dependent cellular cytotoxicity (ADCC) and the production of prostaglandin E2 (PGE2) and superoxide anion (O2-). An assay for the measurement of interleukin-1 (IL-1), based on its ability to induce interleukin-2 (IL-2) production by the EL-4 mouse thymoma line in the presence of the calcium ionophore A23187, was examined. Together, these assays provide the tools to better define drug or chemical effects on the mononuclear phagocyte system (MPS) of this important animal model.

The hematologic effects of cefonicid and cefazedone in the dog: a potential model of cephalosporin hematotoxicity in man.

Cephalosporin antibiotics cause a variety of hematologic disturbances in man, the pathogeneses and hematopathology of which remain poorly characterized. There is a need for a well-defined animal model in which these blood dyscrasias can be studied. In four subacute toxicity studies, the intravenous administration of cefonicid or cefazedone to beagle dogs caused a dose-dependent incidence of anemia, neutropenia, and thrombocytopenia after 1-3 months of treatment. A nonregenerative anemia was the most compromising of the cytopenias and occurred in approximately 50% of dogs receiving 400-500 mg/kg cefonicid or 540-840 mg/kg cefazedone. All three cytopenias were completely reversible following cessation of treatment; the time required for recovery of the erythron (approximately 1 month) was considerably longer than that of the granulocytes and platelets (hours to a few days). Upon rechallenge with either cephalosporin, the hematologic syndrome was reproduced in most dogs tested; cefonicid (but not cefazedone)-treated dogs showed a substantially reduced induction period (15 +/- 5 days) compared to that of the first exposure to the drug (61 +/- 24 days). This observation, along with the rapid rate of decline in red cell mass parameters of affected dogs, suggests that a hemolytic component complicated the red cell production problem and that multiple toxicologic mechanisms contributed to the cytopenia. We conclude that the administration of high doses of cefonicid or cefazedone to dogs can induce hematotoxicity similar to the cephalosporin-induced blood dyscrasias described in man and thus provides a useful model for studying the mechanisms of these disorders.

The hematopathology of cefonicid- and cefazedone-induced blood dyscrasias in the dog.

Cephalosporin treatment in man has been associated with blood dyscrasias that include a time- and dose-related anemia, neutropenia, and thrombocytopenia, the hematopathology of which remains poorly characterized. A similar hematologic syndrome can be produced in dogs following daily intravenous injections of 540-840 mg/kg cefazedone or 400-500 mg/kg cefonicid for 1-3 months. Using this animal model, histologic and cytologic changes in blood, bone marrow, spleen, and liver were studied over the course of the cephalosporin-induced cytopenias. Peripheral blood cytologic observations included an absence, generally, of erythroid regenerative changes, increased numbers of macroplatelets, spherocytosis, erythroblastemia, and toxic neutrophil morphology. Interim and postmortem cytologic and histologic observations of bone marrow included hypoplastic and toxic changes, primarily in cytopenic dogs receiving high doses of cefonicid, and regenerative changes in hematopoietic tissue of affected cefazedone-treated animals. The latter included variable erythroid hyperplasia, increased megakaryocytes, and decreased marrow fat and was accompanied by evidence of extra-medullary hematopoiesis and increased hemosiderin and hemophagocytosis in liver and splenic tissue. The incidence and severity of these changes were dose-dependent, corresponded with the cytopenias observed peripherally, and, like the cytopenias, were fully reversible. These observations suggest that the hematologic syndrome associated with cephalosporin treatment in the dog has multiple toxicologic mechanisms, which include peripheral cytotoxic effects and bone marrow damage with depressed or ineffective hematopoiesis.

The role of conventional pathology and toxicology in evaluating the immunotoxic potential of xenobiotics.

Investigating the immunotoxic potential of candidate drugs as part of a preclinical safety evaluation poses several problems. These include the need for practical, validated tests, the difficulty in establishing the toxicologic significance of positive findings, and a poor understanding of the predictive value such findings hold for drug effects in man. A key component of this investigation is the toxicologic profile generated through preclinical toxicity and safety studies. As this "routine" assessment becomes increasingly comprehensive and sophisticated, most toxicologically significant drug-associated effects are revealed. Such findings may serve as "triggers" for investigating possible immune mechanisms. Decisions to test specifically for immunotoxicity may also be influenced by the molecular structure and pharmacologic profile of the compound, as well as the intended use of the drug. Examples of such indications and follow-up studies are discussed in this review. We are presently poorly equipped to effectively screen drugs indiscriminately for an immunotoxic potential. We are better prepared, however, to investigate whether a drug-associated change is due to an adverse effect on the immune system. This problem-oriented approach to immunotoxicology challenges us as diagnosticians and immunopathologists, and requires a close working relationship among the toxicologic pathologist, the basic immunologist, the immunopharmacologist, and the clinician.

Gold-induced immune thrombocytopenia in the dog.

In two seven-year studies with gold compounds in dogs of both sexes, thrombocytopenia was observed after 45 to 72 months of dosing in three of 14 and two of 14 dogs in high-dose groups that received 2.4 to 3.6 mg/kg of auranofin per day orally or 0.5 to 2.0 mg/kg of gold sodium thiomalate intramuscularly once every three days, respectively. An immune basis for the disorder was suggested by the apparent consumptive nature of the thrombocytopenia (increased bone marrow megakaryocytes and large peripheral blood platelets), the response to corticosteroid therapy and the demonstration of increased platelet-associated immunoglobulin. The latter was demonstrated with a solid phase radioimmunoassay and by electron microscopy using a staphylococcal protein A-colloidal gold conjugate. Platelet-associated immunoglobulin decreased as the platelet counts rose, and in one dog monitored over periods of steroid-induced remissions and subsequent relapses, the amount of platelet-associated immunoglobulin G correlated inversely with the platelet count (r = 0.82). These findings suggest that the long-term administration of gold compounds in dogs is associated with a dose-dependent incidence of thrombocytopenia, which is immune-mediated and similar to that associated with parenteral chrysotherapy in man. The application of tests for platelet-associated immunoglobulin to canine patients with immune thrombocytopenia should be useful in the diagnosis of the disorder in clinical practice.

Eosinophilic myeloproliferative disorder in a horse.

An eosinophilic myeloproliferative disorder resulted in edema and hemorrhagic diathesis in a 10-month-old Standardbred colt. Laboratory abnormalities included severe thrombocytopenia, anemia, mild hypoproteinemia, and marked eosinophilia. Circulating eosinophils were immature or atypical with variation in granule size, disproportionate nuclear to cytoplasmic maturation, and abnormal nuclear size and shape. Bone marrow aspirate had mainly atypical eosinophil precursors, few erythroid precursors, and no megakaryocytes. A blood transfusion and dexamethasone therapy resulted in some improvement; however, the horse was euthanatized due to poor prognosis. Postmortem examination showed gastrointestinal parasitism; histologically the spleen was infiltrated by atypical eosinophils and there were sites of eosinophilopoiesis. The disease was broadly similar to idiopathic hypereosinophilic syndrome in people, but typical cardiac and neurologic involvement of hypereosinophilic syndrome were absent. Progressive myelophthesis and marked eosinophil atypia suggested malignancy.

Effects of Escherichia coli endotoxin on leukocyte and platelet counts, fibrinogen concentrations, and blood clotting in colostrum-fed and colostrum-deficient neonatal calves.

Effects of a single IV injection of Escherichia coli endotoxin on hemogram and clotting function were compared in colostrum-fed and colostrum-deficient neonatal calves. Before endotoxin administration, the 2 groups of calves only differed in their prothrombin times. After endotoxin administration, there were significant differences (P less than 0.005) between colostrum-fed and colostrum-deficient calves in total leukocyte, segmented neutrophil, nonsegmented neutrophil, and lymphocyte (P less than 0.05) counts and partial thromboplastin time. Significant time dependent changes were observed in the aforementioned parameters and in platelet count and fibrinogen concentration. Seemingly, colostrum feeding improved the calf's ability to respond to endotoxin challenge exposure probably because of improved granulopoietic activity.

Changes in selected biochemical constituents of blood collected from horses participating in a 50-mile endurance ride.

The effects of strenuous exercise on serum electrolytes, blood metabolites, and serum enzymes were studied in a group of 13 horses participating in a 50-mile endurance ride. Blood samples were collected before, during, and at the end of the ride, as well as 1 hour and 16 hours after the completion of the ride. There were significant changes in these values when preride values were compared with those of samples taken at different sample-collection periods. Significant (P less than 0.001) decreases were observed in serum concentrations of chloride, potassium, and calcium. A significant increase in the serum concentration of phosphorous also was observed. The mean concentrations of chloride and calcium remained significantly reduced in the 16-hour postride sample. A small, but significant (P less than 0.05), increase in sodium and decrease in glucose concentrations were observed at the midride sample. Although midride samples did not reflect a significant change in blood pyruvate concentration, a significant (P less than 0.05) increase was observed at the end of the ride. Changes in the hematocrit and the total protein concentration were statistically significant at levels of P less than 0.001 and P less than 0.01, respectively. The horses showed highly significant increases in concentrations of free fatty acids, serum enzymes (P less than 0.001), and lactate (P less than 0.005) during the ride. The elevated values of creatine kinase and lactic dehydrogenase decreased, while the aspartate aminotransferase values remained increased long after completion of the ride. The lactate and free fatty acids, which rose significantly during the ride, decreased to approximately preride values by 16 hours after the ride was completed. We conclude that the changes in serum electrolytes, enzymes, and blood metabolites reflect the sweating, muscle damage, and increased dependency on anaerobic glycolysis and lipid mobilization that these horses experienced during the competitive endurance ride.

Glucocorticoid receptors in peripheral blood lymphocytes from bovine leukemia virus-infected cows with persistent lymphocytosis.

Bovine leukemia virus-infected cows with persistent lymphocytosis have an expanded population of B-lymphocytes in the peripheral blood that is sensitive to glucocorticoids in vitro and in vivo. We examined peripheral blood lymphocytes from cows with persistent lymphocytosis for the presence of specific glucocorticoid receptors. Steroid binding in intact cells was determined by a whole cell competitive binding assay using [3H]dexamethasone. The binding of the glucocorticoid to receptor was characterized in terms of affinity, specificity, and kinetics of the reaction. We found that peripheral blood lymphocytes from three cows with persistent lymphocytosis had 5000 to 6600 specific glucocorticoid-binding sites/cell. Compared with that reported for human lymphoid cells, glucocorticoid receptors in the bovine lymphocytes were found to have a greater affinity for the steroid with an association rate that was three times faster and a dissociation rate that was less than one-half of the former. We examined the biological half-life of hydrocortisone in the normal cow and found it to be 69.3 min, which is shorter than that reported for other domestic species and humans. The kinetics and affinity of the steroid binding may explain why in vivo glucocorticoid sensitivity was demonstrated in these animals despite the fact that elevated levels of plasma corticoids were not maintained. These results suggest that glucocorticoid sensitivity may be influenced by the nature of the binding reaction between steroid and receptor.

Glucocorticoid effects on peripheral blood lymphocytes in cows infected with bovine leukemia virus.

We examined the effects of glucocorticoids on peripheral blood lymphocytes (PBL) in lymphoproliferative conditions associated with bovine leukemia virus (BLV): persistent lymphocytosis (PL) and lymphosarcoma cell leukemia (BLSL). The effects of hydrocortisone 21-sodium succinate (HSS) on spontaneous incorporation (SI) and mitogen-stimulated incorporation of radiolabeled-thymidine and the effects of intramuscular administration of prednisolone acetate were studied. An expanded population of B lymphocytes in cows with PL was remarkable sensitive to glucocorticoids in vitro and in vivo. SI was markedly inhibited by concentrations of HSS as low as 10(-7) M. These results correlated well with in vivo observations, where an 80%-90% decrease in PBL occurred during the course of glucocorticoid administration. The decrease in total lymphocytes was accounted for almost entirely by a decrease in the expanded B lymphocyte population. Steroid-sensitive lymphocytes together with steroid-resistant cells were observed in cows with BLSL. The reduction in the steroid-sensitive lymphocytes was associated with rapid disease progression in cows with lymphosarcoma. Steroid-sensitive lymphocyte populations in cows with BLSL may include the same reactive B-cell population found in cows with PL. Glucocorticoids may prove to be a useful tool for study of the immune response to the oncogenic virus and lymphoma in BLV-infected cattle.