Preliminary Study on Safety Conditions in Health Workers Exposed to Antineoplastic Drugs

A study was done to establish work practices and preventive measures for nurses handling antineoplasticdrugs (AND) and to determine the risk of developing AND-related symptoms. A descriptive cross sectional study was made. Workers from 5 health centers in Valencia, Venezuela, were selected. Demography, occupational and clinical history, shift, work practices, safety precautions, antineoplastic drugs used, residues disposal and life styles were obtained via a questionnaire. Most prevalent symptoms were adjusted for age, shift, and smoking. Age was significant for cough and dizziness; smoking was significant for abdomipo seleccionado de trabajadores que manejan FAN en cinco centros de salud de la ciudad de Valencia, Venezuela, con el propósito de evaluar de forma preliminar su riesgo potencial de desarrollar signos-síntomas derivados de este oficio; a su vez, establecer si existe la necesidad de continuar con una evaluación más profunda que permita hacer recomendaciones concluyentes para evitar sus efectos adversos. Metodología La población estuvo constituida por el personal de las unidades de oncología de cinco centros de salud de la ciudad de Valencia, Venezuela. La muestra la conformaron veinte trabajadores que pertenecen a dichos centros. La participación fue voluntaria y mediante firma de una carta de consentimiento. Recolección de datos Se realizó una entrevista a cada participante con información que hizo referencia a datos demográficos, historia ocupacional, turnos de trabajo, entrenamiento en medidas de seguridad para el manejo de este tipo de fármacos, actividades que realizan, uso de equipos de protección personal (EPP), sitio de manipulación de nal pain and shift was significant for nausea and cough. Nauseas were the most prevalent symptom (55%). Dizziness was directly associated with use of gowns and inversely half-face respirator. None of the studied centers had satisfactory working conditions. A follow up study should be made including physical exam and environmental and biological monitoring.

Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Acute phase proteins in canine lymphoma during antineoplastic chemotherapy

The lymphoma is the main hematopoietic tumor in dogs and it is characterized by the proliferation of cells from lymphoid tissue, histiocytes and its precursors. Animals with lymphoma often show changes in biochemical and hematological parameters such as non-regenerative normochromic normocytic anemia, hemolytic anemia, hypocalcaemia and monoclonal gammopathy. The development of tumor can cause alterations in serum concentrations of acute phase proteins (APPs), consequent of hepatocytes stimulus by cytokines of inflammatory action. This study aimed to quantify and qualify APPs in dogs with lymphoma, at diagnosis time and during the time of chemotherapy sessions. After syneresis, centrifugation and fractioning the serum samples of 10 healthy and 10 dogs with lymphomas, the proteins fractions were separated by polyacrilamide gel electrophoresis (SDS-PAGE) and its concentrations were determined by computer densitometry. Between 18 and 30 proteins were separated by eletrophoresis, with molecular weights ranging from 18 to 245 kDa (kilodaltons). The alpha-1-glicoprotein acid (AGP) and transferrin serum concentration showed significantly higher in dogs with lymphoma, when compared with healthy dogs at diagnosis. The alpha-1-antitripsin (AAT) serum concentrations showed significantly higher in healthy dogs, when compared with dogs with lymphoma at diagnosis. The dogs with lymphoma the albumin did not appear as negative APP. On the other hand, transferrin appeared as positive AAP at diagnosis time and during the chemotherapy sessions. Healthy dogs had AAT serum concentrations significantly higher when compared to dogs with lymphoma at diagnosis. So, in this trial, it is suggested that this protein has been shown as a negative APP in the dogs with lymphoma. These dogs presented significantly higher AGP serum concentrations, in relation to healthy dogs at diagnosis, evidencing this protein APP positive behavior in neoplasm.

Dental anomalies in children submitted to antineoplastic therapy

Cancer is the third most frequent cause of death in children in Brazil. Early diagnosis and medical advances have significantly improved treatment outcomes, which has resulted in higher survival rates and the management of late side effects has become increasingly important in caring for these patients. Dental abnormalities are commonly observed as late effects of antineoplastic therapy in the oral cavity. The incidence and severity of the dental abnormalities depend on the child's age at diagnosis and the type of chemotherapeutic agent used, as well as the irradiation dose and area. The treatment duration and aggressivity should also be considered. Disturbances in dental development are characterized by changes in shape, number and root development. Enamel anomalies, such as discoloration, opacities and hypoplasia are also observed in these patients. When severe, these abnormalities can cause functional and esthetic sequelae that have an impact on the children's and adolescents' quality of life. General dentists and pediatric dentists should understand these dental abnormalities and how to identify them aiming for early diagnosis and appropriate treatment.

Sister chromatid exchanges and chromosome aberrations in lymphocytes of nurses handling antineoplastic drugs

Chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) were investigated in peripheral lymphocytes of 15 nurses and nurse's aides handling cytostatic agents in hospital oncology units. Significantly increased frequencies were noted for both CA and SCE rates when the exposed individuals were compared with 15 nurses working in other hospital units and to a control sample matched by sex and age. This points to the need for emphasizing protective measures in the handling of anti-neoplastic agents.

Cytogenetic assessment of occupational exposure to antineoplastic agents

This study has evaluated the utility of measuring effects of low level occupational exposure of nursing personnel to antineoplastic agents. The effect measured in this study is chromosomal damage in peripheral lymphocytes (chromosomal breakage and micronuclei frequency).^ Using nursing personnel in three exposure classifications (low, moderate and high) and breast cancer patients before and after treatment with antineoplastic agents, a weak but statistically significant association was found between exposure and chromosomal damage. Of special interest was the finding that consistent glove usage was negatively associated with increased chromosomal damage.^ The study also demonstrated a statistically significant association between the two measures of chromosomal damage: chromosomal breakage and micronuclei frequency. This suggests that the micronucleus method is a useful test for studying cytogenetic effects in lymphocytes. ^

INACTIVATION OF MUTAGENIC DRUGS AND THEIR METABOLITES IN THE URINE OF PATIENTS ADMINISTERED ANTINEOPLASTIC THERAPY

Urines from patients administered mutagenic antineoplastic drugs were significantly mutagenic in the Ames assay, and hence may pose a genotoxic hazard to hospital personnel or family members caring for the patient. The urines were tested for mutagenicity in several different strains of Salmonella typhimurium that were uvr positive or negative (TA98, TA100, TA102, UTH8413, UTH8414). The urines were fractionated by high pressure liquid chromatography (HPLC) and the fractions assayed for mutagenicity in the strains in which the whole urine was mutagenic. Only fractions of urines containing the parent compound (cisplatin, doxorubicin, or mitomycin) were mutagenic; no other fraction showed significant mutagenicity. However, urine containing cyclophosphamide had two fractions that were mutagenic. One fraction, the fraction containing cyclophosphamide, required metabolic activation for mutagenicity. The other fraction did not require activation for mutagenicity.^ The chemical and mutagenic stability of these urines at room temperature was assayed over a 14 day period. The parent compound degraded within the first seven days, but the urines remained mutagenic. Cis-platinum was chemically stable in the urine; however, the urine decreased in mutagenicity. The decrease was probably the result of stable ligands binding to the platinum.^ Inactivation methods were developed to reduce the genotoxic hazard. Urine containing cisplatin was inactivated by complexing the cisplatin with diethyldithiocarbamate (DDTC). Oxidation with NaOCl of urines containing mitomycin and doxorubicin (sodium thiosulfate must be added to the doxorubicin urine) results in mutagenic inactivation. Inactivation of urine containing cyclophosphamide requires oxidation with alkaline potassium permaganate and trapping of active degradation products with sodium thiosulfate. Urines containing these drugs can be inactivated, but not always by the same method that inactivates the drug alone in solution. Therefore, in the future development of inactivation methods, both chemical and mutagenic assays are necessary to determine effectiveness. Methods of inactivation of mutagenic excreta developed in this study are both effective and practical. ^

EXPOSURE OF PHARMACY PERSONNEL TO ANTINEOPLASTIC DRUGS

This dissertation consists of two parts: (1) Exposure of pharmacy personnel to antineoplastic drugs. The Salmonella reversion test was used to measure the mutagenic activities of urine concentrates from individuals preparing antineoplastic drugs for intravenous administration. Longitudinal studies were performed in which the total urine produced in 24-hour periods was collected, starting on a Sunday at 7 P.M. after a duty-free weekend and extending over an eight-day period. There was no detectable increase in mutagenic activity in the urine concentrates of three pharmacy administrators who had no contact with these drugs. All six individuals admixing drugs in open-faced, horizontal laminar flow hoods displayed a two-fold increase in mutagenesis by the fourth day with peak values of 2.7 to 24-fold occurring on days five and six, reduced values by day seven with a return to the spontaneous level by day eight. When four of the six positive individuals in the preceding experiment admixed comparable amounts of antineoplastic drugs in a closed-faced, vertical laminar flow hood, no increase in mutagenic activity was detected in their urine concentrates over the eight-day period. (2) Estimate of potential carcinogenic risks of antineoplastic drugs. Excision repair is the major repair system that is involved with the elimination of chemically induced DNA (deoxyribonucleic acid) lesions. This DNA excision repair capability increases in mammalian species with longer life span such as humans. In this study, the effect of functional DNA excision repair on the mutagenesis invoked by 17 antineoplastic drugs was determined by using a Salmonella/Microsome assay which was expanded to include some uvr('+) counterparts of the excisionless (uvrB) tester strains routinely employed. Although extrapolation cannot be made from bacteria to humans, one should be able to make a qualitative comparison as to which antineoplastic drugs are more potentially carcinogenic to humans based on the effects of excision repair on their mutagenesis in bacteria. The tested antineoplastic drugs were divided into three classes: those requiring excision repair for mutagenesis; those producing nonrepairable genetic damage; and those producing mostly repairable premutational DNA lesions. ^

Increased geranylgeranylated K-Ras contributes to antineoplastic effects of farnesyltransferase inhibitors.

The Ras family of small GTPases (N-, H-, and K-Ras) is a group of important signaling mediators. Ras is frequently activated in some cancers, while others maintain low level activity to achieve optimal cell growth. In cells with endogenously low levels of active Ras, increasing Ras signaling through the ERK and p38 MAPK pathways can cause growth arrest or cell death. Ras requires prenylation – the addition of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) group – to keep the protein anchored into membranes for effective signaling. N- and K-Ras can be alternatively geranylgeranylated (GG’d) if farnesylation is inhibited but are preferentially farnesylated. Small molecule inhibitors of farnesyltransferase (FTIs) have been developed as a means to alter Ras signaling. Our initial studies with FTIs in malignant and non-malignant cells revealed FTI-induced cell cycle arrest, reduced proliferation, and increased Ras signaling. These findings led us to the hypothesis that FTI induced increased GG’d Ras. We further hypothesized that the specific effects of FTI on cell cycle and growth result from increased signal strength of GG’d Ras. Our results did show that increase in GG’d K-Ras in particular results in reduced cell viability and cell cycle arrest. Genetically engineered constructs capable of only one type of prenylation confirmed that GG’d K-Ras recapitulated the effect of FTI in 293T cells. In tumor cell lines ERK and p38 MAPK pathways were both strongly activated in response to FTI, indicating the increased activity of GG’d K-Ras results in antiproliferative signals specifically through these pathways. These results collectively indicate FTI increases active GG’d K-Ras which activates ERK and p38 MAPKs to reduced cell viability and induce cell cycle arrest in malignant cells. This is the first report that identifies increased activity of GG’d K-Ras contributes to antineoplastic effects from FTI by increasing the activity of downstream MAPKs. Our observations suggest increased GG’d K-Ras activity, rather than inhibition of farnesylated Ras, is a major source of the cytostatic and cytotoxic effects of FTI. Our data may allow for determination of which patients would benefit from FTI by excluding tumors or diseases which have strong K-Ras signaling.

TRAP220 is modulated by the antineoplastic agent 6-Mercaptopurine, and mediates the activation of the NR4A subgroup of nuclear receptors

The NR4A1-3 (Nur77, NURR1 and NOR-1) subfamily of nuclear hormone receptors (NRs) has been implicated in Parkinson's disease, schizophrenia, manic depression, atherogenesis, Alzheimer's disease, rheumatoid arthritis, cancer and apoptosis. This has driven investigations into the mechanism of action, and the identification of small molecule regulators, that may provide the platform for pharmaceutical and therapeutic exploitation. Recently, we found that the purine antimetabolite 6-Mercaptopurine (6-MP), which is widely used as an anti-neoplastic and anti-inflammatory drug, modulated the NR4A1-3 subfamily. Interestingly, the agonist-mediated activation did not involve modulation of primary coactivators' (e.g. p300 and SRC-2/GRIP-1) activity and/or recruitment. However, the role of the subsequently recruited coactivators, for example CARM-1 and TRAP220, in 6-MP-mediated activation of the NR4A1-3 subfamily remains obscure. In this study we demonstrate that 6-MP modulates the activity of the coactivator TRAP220 in a dose-dependent manner. Moreover, we demonstrate that TRAP220 potentiates NOR-1-mediated transactivation, and interacts with the NR4A1-3 subgroup in an AF-1-dependent manner in a cellular context. The region of TRAP220 that mediated 6-MP activation and NR4A interaction was delimited to amino acids 1-800, and operates independently of the critical PKC and PKA phosphorylation sites. Interestingly, TRAP220 expression does not increase the relative induction by 6-MP, however the absolute level of NOR-1-mediated trans-activation is increased. This study demonstrates that 6-MP modulates the activity of the NR4A subgroup, and the coactivator TRAP220.

Antineoplastic Cytotoxicity and Immune Adjuvancy of a Recombinant Oncolytic Poliovirus

Our group has pioneered the development of a live-attenuated poliovirus, called PVSRIPO, for the purpose of targeting cancer. Despite clinical progress, the cancer selective cytotoxicity and immunotherapeutic potential of PVSRIPO has not yet been mechanistically dissected. Defining such mechanisms may inform its clinical application.

Herein I describe the discovery of a mechanism by which the MAP-Kinase Interacting Kinases (MNKs) regulate PVSRIPO cytotoxicity in cancer. In doing so, I delineate a novel, intricate network connecting the MNK and mTOR signaling pathway that regulates activity of a splicing kinase called the Ser-Arg Rich Protein Kinase (SRPK), and define SRPK as an impediment to IRES mediated translation. Moreover, I demonstrate that MNK regulates mTORC1 associations that determine its substrate proximity and thus, activity. In a collaborative effort, we found that PVSRIPO oncolysis produces antigen specific, cytolytic anti-tumor immunity in an in vitro human system and that much of the observed adjuvancy is due to the direct infection of dendritic cells (DCs) by the virus itself; implicating PVSRIPO as a potent adjuvant. In summary, oncogenic signaling in part through MNK leads to cancer specific cytotoxicity by PVSRIPO that engages an inflammatory environment conducive to DC activation and antigen specific T cell antigen immunity.