Expression of Phosphorylated KIT in Canine Mast Cell Tumor.

Canine cutaneous mast cell tumor (MCT) is the most common canine skin tumor and exhibits variable biologic behavior. Signaling through the KIT receptor tyrosine kinase promotes cellular proliferation and survival and has been shown to play a role in MCT progression. Despite investigations into numerous biomarkers and the proposal of several grading schemas, no single marker or grading system can accurately predict outcome in canine MCT. The first aim of this study was to develop an immunohistochemical assay to measure phosphorylated KIT (pKIT) to investigate its association with 2 commonly used grading systems and other established prognostic markers for canine MCT. Thirty-four archived MCTs were evaluated for expression of pKIT and Ki-67, KIT localization, mitotic count, mutations in exons 8 and 11 in c-kit, and grading by the Patnaik and 2-tier systems. Expression of pKIT was significantly ( P < .05) correlated with the 2-tier grading scheme and c-kit mutation. Correlation approached significance ( P = .06) with Mitotic Index (MI) and Ki-67. An additional aim was to determine whether pKIT labeling provides a pharmacodynamic marker for predicting response to the receptor tyrosine kinase inhibitor toceranib (TOC). MCTs from 4 of 7 patients demonstrated a partial response to TOC. pKIT expression was assessed by immunohistochemistry in biopsies obtained before and 6 hours after the patients were treated with TOC. Reduced pKIT expression after TOC treatment was demonstrated in 3 of the 4 patients with a partial response compared to 1 of the 3 nonresponders. Collectively, these results demonstrate that immunohistochemical detection of pKIT may be a clinically relevant assay to evaluate the activation status of the major oncogenic pathway in canine MCT.

The Flint Animal Cancer Center (FACC) Canine Tumour Cell Line Panel: a resource for veterinary drug discovery, comparative oncology and translational medicine.

Mammalian cell tissue culture has been a critical tool leading to our current understanding of cancer including many aspects of cellular transformation, growth and response to therapies. The current use of large panels of cell lines with associated phenotypic and genotypic information now allows for informatics approaches and in silico screens to rapidly test hypotheses based on simple as well as complex relationships. Current cell line panels with large amounts of associated drug sensitivity and genomics data are comprised of human cancer cell lines (i.e. NCI60 and GDSC). There is increased recognition of the contribution of canine cancer to comparative cancer research as a spontaneous large animal model with application in basic and translational studies. We have assembled a panel of canine cancer cell lines to facilitate studies in canine cancer and report here phenotypic and genotypic data associated with these cells.

Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer.

Aberrant T-cell factor (TCF) transcription is implicated in the majority of colorectal cancers (CRCs). TCF transcription induces epithelial-mesenchymal transition (EMT), promoting a tumor-initiating cell (TIC) phenotype characterized by increased proliferation, multidrug resistance (MDR), invasion and metastasis. The data presented herein characterize topoisomerase IIα (TopoIIα) as a required component of TCF transcription promoting EMT. Using chromatin immunoprecipitation (ChIP) and protein co-immunoprecipitation (co-IP) studies, we show that TopoIIα forms protein-protein interactions with ß-catentin and TCF4 and interacts with Wnt response elements (WREs) and promoters of direct target genes of TCF transcription, including: MYC, vimentin, AXIN2 and LEF1. Moreover, both TopoIIα and TCF4 ChIP with the N-cadherin promoter, which is a new discovery indicating that TCF transcription may directly regulate N-cadherin expression. TopoIIα N-terminal ATP-competitive inhibitors, exemplified by the marine alkaloid neoamphimedine (neo), block TCF activity in vitro and in vivo. Neo effectively inhibits TopoIIα and TCF4 from binding WREs/promoter sites, whereas protein-protein interactions remain intact. Neo inhibition of TopoIIα-dependent TCF transcription also correlates with significant antitumor effects in vitro and in vivo, including the reversion of EMT, the loss of TIC-mediated clonogenic colony formation, and the loss of cell motility and invasion. Interestingly, non-ATP-competitive inhibitors of TopoIIα, etoposide and merbarone, were ineffective at preventing TopoIIα-dependent TCF transcription. Thus, we propose that TopoIIα participation in TCF transcription may convey a mechanism of MDR to conventional TopoIIα inhibitors. However, our results indicate that TopoIIα N-terminal ATP-binding sites remain conserved and available for drug targeting. This article defines a new strategy for targeted inhibition of TCF transcription that may lead to effective therapies for the treatment of CRC and potentially other Wnt-dependent cancers.

Limited sampling pharmacokinetics of subcutaneous ondansetron in healthy geriatric cats, cats with chronic kidney disease, and cats with liver disease.

Ondansetron, a 5-HT3 receptor antagonist, is an effective anti-emetic in cats. The purpose of this study was to compare pharmacokinetics of subcutaneous (SQ) ondansetron in healthy geriatric cats to cats with chronic kidney disease (CKD) or liver disease using a limited sampling strategy. 60 cats participated; 20 per group. Blood was drawn 30 and 120 min following one 2 mg (mean 0.49 mg/kg, range 0.27-1.05 mg/kg) SQ dose of ondansetron. Ondansetron concentrations were measured by liquid chromatography coupled to tandem mass spectrometry. Drug exposure represented as area under the curve (AUC) was predicted using a limited sampling approach based on multiple linear regression analysis from previous full sampling studies, and clearance (CL/F) estimated using noncompartmental methods. Kruskal-Wallis anova was used to compare parameters between groups. Mean AUC (ng/mL·h) of subcutaneous ondansetron was 301.4 (geriatric), 415.2 (CKD), and 587.0 (liver). CL/F (L/h/kg) of SQ ondansetron was 1.157 (geriatric), 0.967 (CKD), and 0.795 (liver). AUC was significantly higher in liver and CKD cats when compared to geriatric cats (P < 0.05). CL/F in liver cats was significantly decreased (P < 0.05) compared to geriatric cats. In age-matched subset analysis, AUC and CL/F in liver cats remained significantly different from geriatric cats.

Single- and multiple dose pharmacokinetics and multiple dose pharmacodynamics of oral ABT-116 (a TRPV1 antagonist) in dogs.

Six dogs were used to determine single and multiple oral dose pharmacokinetics of ABT-116. Blood was collected for subsequent analysis prior to and at 15, 30 min and 1, 2, 4, 6, 12, 18, and 24 h after administration of a single 30 mg/kg dose of ABT-116. Results showed a half-life of 6.9 h, k(el) of 0.1/h, AUC of 56.5 µg·h/mL, T(max) of 3.7 h, and C(max) of 3.8 µg/mL. Based on data from this initial phase, a dose of 10 mg/kg of ABT-116 (no placebo control) was selected and administered to the same six dogs once daily for five consecutive days. Behavioral observations, heart rate, respiratory rate, temperature, thermal and mechanical (proximal and distal limb) nociceptive thresholds, and blood collection were performed prior to and 4, 8, and 16 h after drug administration each day. The majority of plasma concentrations were above the efficacious concentration (0.23 µg/mL previously determined for rodents) for analgesia during the 24-h sampling period. Thermal and distal limb mechanical thresholds were increased at 4 and 8 h, and at 4, 8, and 16 h respectively, postdosing. Body temperature increased on the first day of dosing. Results suggest adequate exposure and antinociceptive effects of 10 mg/kg ABT-116 following oral delivery in dogs.

Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma.

The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer-related signalling pathways despite differences in activating mutations.

Oral, subcutaneous, and intravenous pharmacokinetics of ondansetron in healthy cats.

Ondansetron is a 5-HT3 receptor antagonist that is an effective anti-emetic in cats. The purpose of this study was to evaluate the pharmacokinetics of ondansetron in healthy cats. Six cats with normal complete blood count, serum biochemistry, and urinalysis received 2 mg oral (mean 0.43 mg/kg), subcutaneous (mean 0.4 mg/kg), and intravenous (mean 0.4 mg/kg) ondansetron in a cross-over manner with a 5-day wash out. Serum was collected prior to, and at 0.25, 0.5, 1, 2, 4, 8, 12, 18, and 24 h after administration of ondansetron. Ondansetron concentrations were measured using liquid chromatography coupled to tandem mass spectrometry. Noncompartmental pharmacokinetic modeling and dose interval modeling were performed. Repeated measures anova was used to compare parameters between administration routes. Bioavailability of ondansetron was 32% (oral) and 75% (subcutaneous). Calculated elimination half-life of ondansetron was 1.84 ± 0.58 h (intravenous), 1.18 ± 0.27 h (oral) and 3.17 ± 0.53 h (subcutaneous). The calculated elimination half-life of subcutaneous ondansetron was significantly longer (P < 0.05) than oral or intravenous administration. Subcutaneous administration of ondansetron to healthy cats is more bioavailable and results in a more prolonged exposure than oral administration. This information will aid management of emesis in feline patients.

Development of a limited-sampling model for prediction of doxorubicin exposure in dogs.

Understanding the relationship between drug dose and exposure (pharmacokinetics, PK) and the relationship between exposure and effect (pharmacodynamics) is an important component of pharmacology when attempting to predict clinical effects of anticancer drugs. PK studies can provide a better understanding of these relationships; however, they often involve intensive sampling over an extended period of time, resulting in increased cost and decreased compliance. Doxorubicin (DOX), one of the most widely used antineoplastic agents in veterinary cancer therapy, is characterized by large interpatient variability in overall drug exposure and the development and degree of myelosuppression following equivalent dosages. We have developed and validated a limited-sampling strategy for DOX, in which three blood samples are obtained over 1 h post-treatment, that accurately predicts patient exposure. This strategy could allow for refining of dosing variables and utilization of therapeutic drug monitoring to ensure optimized dosing.

Milk thistle and its derivative compounds: a review of opportunities for treatment of liver disease.

Milk thistle extracts have been used as a "liver tonic" for centuries. In recent years, silibinin, the active ingredient in milk thistle extracts, has been studied both in vitro and in vivo to evaluate the beneficial effects in hepatic disease. Silibinin increases antioxidant concentrations and improves outcomes in hepatic diseases resulting from oxidant injury. Silibinin treatment has been associated with protection against hepatic toxins, and also has resulted in decreased hepatic inflammation and fibrosis. Limited information currently is available regarding silibinin use in veterinary medicine. Future study is justified to evaluate dose, kinetics, and treatment effects in domestic animals.

The pharmacokinetics of mirtazapine in cats with chronic kidney disease and in age-matched control cats.

BACKGROUND: Cats with chronic kidney disease (CKD) often experience inappetence, and may benefit from administration of mirtazapine, an appetite stimulant. The pharmacokinetics of mirtazapine in CKD cats is unknown. HYPOTHESIS: CKD delays the clearance/bioavailability (CL/F) of mirtazapine. ANIMALS: Six CKD cats and 6 age-matched controls (AMC) were enrolled. Two CKD cats each from International Renal Interest Society (IRIS) stage II, III and IV were included. METHODS: Blood samples were collected before and 0.5, 1, 1.5, 2, 4, 8, 24, and 48 hours after a single PO dose of 1.88 mg of mirtazapine. Mirtazapine concentrations were measured by liquid chromatography coupled to tandem mass spectrometry. Non-compartmental pharmacokinetic modeling was performed. RESULTS: Mean age was 11 years (CKD cats) and 10.8 years (AMC cats). Mean serum creatinine concentration ± standard deviation (SD) was 3.8 ± 1.6 mg/dL (CKD) and 1.3 ± 0.4 mg/dL (AMC). Mean half-life ± SD was 15.2 ± 4.2 hours (CKD) and 12.1 ± 1.1 hours (AMC). Mean area under the curve (AUC) ± SD was 770.6 ± 225.5 ng/mL•hr (CKD) and 555.5 ± 175.4 ng/mL•hr (AMC). Mean CL/F ± SD was 0.6 ± 0.1 L/hr/kg (CKD) and 0.8 ± 0.16 L/hr/kg (AMC). A Mann-Whitney test indicated statistically significant differences in AUC (P = 0.01) and CL/F (P = 0.04) between groups. Calculated accumulation factor for 48-hour dosing in CKD cats was 1.15. CONCLUSION: CKD may delay the CL/F of mirtazapine. A single low dose of mirtazapine resulted in a half-life compatible with a 48-hour dosing interval in CKD cats.

Evaluation of satraplatin in dogs with spontaneously occurring malignant tumors.

BACKGROUND: Satraplatin is the 1st orally bioavailable platinum anticancer drug. OBJECTIVE: Our objectives were to evaluate efficacy in vitro against a canine cancer cell line, to determine the maximally tolerated dose (MTD) of satraplatin in tumor-bearing dogs, to identify the dose-limiting and other toxicities in dogs, and to record pharmacokinetics (PK). ANIMALS: Dogs with macro- or microscopic malignant neoplasia. METHODS: D17 canine osteosarcoma cells first were evaluated in a clonogenic survival assay. Then, dogs with a diagnosis of malignant neoplasia were prospectively entered in standard 3 + 3 cohorts. Additional patients were entered at the MTD to assess efficacy. Total and free platinum (by ultrafiltrate) concentrations were determined with inductively coupled plasma mass spectroscopy. RESULTS: Satraplatin inhibited clonogenic survival in vitro at clinically relevant and achievable concentrations. Twenty-three dogs were treated, 14 with PK evaluation. The MTD was 35 mg/m(2)/d for 5 days, repeated every 3-4 weeks. Bioavailability was 41%. PK variables (mean ± SD) at the MTD included T(max) 1.8 (± 0.7) hours, C(max) 72 (± 26) ng/mL, area under concentration (AUC)(0-24 h) 316 (± 63) h × ng/mL, and MRT 7 (± 1.3) hours. Higher AUC after the 5th versus the 1st dose suggested drug accumulation. Interestingly, platelets consistently reached nadir sooner than did neutrophils (day 14 versus 19). Myelosuppression was dose-limiting and gastrointestinal toxicity was mild. CONCLUSIONS AND CLINICAL IMPORTANCE: Satraplatin was well tolerated in tumor-bearing dogs, thus warranting further investigation in a phase II trial.

Pharmacokinetics of cyclophosphamide after oral and intravenous administration to dogs with lymphoma.

BACKGROUND: Cyclophosphamide is an alkylating chemotherapeutic drug administered i.v. or p.o.. It is currently assumed that exposure to the active metabolite, 4-hydroxycyclophosphamide (4-OHCP), is the same with either route of administration. OBJECTIVES: To characterize the pharmacokinetics of cyclophosphamide and 4-OHCP in dogs with lymphoma when administered p.o. or i.v.. ANIMALS: Sixteen client-owned dogs with substage A lymphoma were enrolled in the study. Eight dogs received cyclophosphamide i.v. and 8 received it p.o.. METHODS: Prospective randomized clinical trial was performed. Blood was collected from each dog at specific time points after administration of cyclophosphamide. The serum was evaluated for the concentration of cyclophosphamide and 4-OHCP with mass spectrometry and liquid chromatography. RESULTS: Drug exposure to cyclophosphamide measured by area under the curve (AUC)(0-inf) is significantly higher after intravenous administration (7.14 ± 3.77 µg/h/mL) compared with exposure after oral administration (P-value < .05). No difference in drug exposure to 4-OHCP was detected after i.v. (1.66 ± 0.36 µg/h/mL) or p.o. (1.42 ± 0.64 µg/h/mL) administered cyclophosphamide. CONCLUSIONS AND CLINICAL IMPORTANCE: Drug exposure to the active metabolite 4-OHCP is equivalent after administration of cyclophosphamide either p.o. or i.v..

Induction of VEGF by tepoxalin does not lead to increased tumour growth in a canine osteosarcoma xenograft.

The purpose of this study was to determine the impact of the non-steroidal anti-inflammatory drug tepoxalin on canine tumour cell growth and describe the changes associated with tepoxalin treatment. In vitro experiments were performed to assess tepoxalin-associated alterations in tumour cell growth. Clinically achievable tepoxalin concentrations did not significantly alter tumour cell growth in vitro. Vascular endothelial growth factor (VEGF) production and hypoxia-inducible factor-1α dose-dependently increased in vitro in the presence of tepoxalin. A canine osteosarcoma xenograft was used to determine in vivo effects of tepoxalin on tumour growth and angiogenesis. Despite increased VEGF in vitro, there was a significant growth delay associated with tepoxalin treatment. Normal dogs were administered tepoxalin to assess effects on systemic VEGF production, but not found to have significantly increased VEGF. These data suggest that tepoxalin may moderately inhibit tumour growth and may be administered as an analgesic to tumour-bearing dogs.

Studies on the pharmacokinetics and pharmacodynamics of mirtazapine in healthy young cats.

Mirtazapine pharmacokinetics was studied in 10 healthy cats. Blood was collected before, and at intervals up to 72 h after, oral dose of 3.75 mg (high dose: HD) or 1.88 mg (low dose: LD) of mirtazapine. Liquid chromatography coupled to tandem mass spectrometry was used to measure mirtazapine, 8-hydroxymirtazapine and glucuronide metabolite concentrations. Noncompartmental pharmacokinetic modeling was performed. Median half-life was 15.9 h (HD) and 9.2 h (LD). Using Mann-Whitney analysis, a statistically significant difference between the elimination half-life, clearance, area under the curve (AUC) per dose, and AUC(∞) /dose of the groups was found. Mirtazapine does not appear to display linear pharmacokinetics in cats. There was no significant difference in glucuronidated metabolite concentration between groups. Pharmacodynamics was studied in 14 healthy cats administered placebo, LD and HD mirtazapine orally once in a crossover, blinded trial. In comparison with placebo, cats ingested significantly more food when mirtazapine was administered. No difference in food ingestion was seen between HD and LD, but significantly more behavior changes were seen with the HD. Limited serum sampling during the pharmacodynamic study revealed drug exposure comparable with the pharmacokinetic study, but no correlation between exposure and food consumed. Mirtazapine (LD) was administered daily for 6 days with no drug accumulation detected.

Safety and efficacy of the combination of erlotinib and sirolimus for the treatment of metastatic renal cell carcinoma after failure of sunitinib or sorafenib.

BACKGROUND: The mammalian target of rapamycin (mTOR) is an important therapeutic target in the treatment of renal cell carcinoma (RCC). Pre-clinical data indicate that the combined inhibition of both the epidermal growth factor receptor and mTOR results in enhanced anticancer activity. METHODS: All patients had metastatic RCC with progression after treatment with sunitinib and/or sorafenib. Treatment consisted of erlotinib 150 mg orally once a day starting on day 1 and sirolimus 6 mg orally on day 8 followed by 2 mg daily, adjusted according to blood levels. RESULTS: A total of 25 patients were enrolled between July 2006 and March 2008. The median progression-free survival (PFS) was 12 weeks (95% CI 5.9-18.1) and median overall survival (OS) 40 weeks (95% CI 0-85.7). No confirmed complete or partial responses were observed, but stable disease >6 months was noted in 21.8% (95% CI 4.9-38.6) of patients. The most common adverse events were rash and diarrhoea. There was no correlation between erlotinib, OSI-420 (days 8 and 15) or sirolimus (days 15 and 29) blood levels and PFS or OS. CONCLUSIONS: The combination of sirolimus and erlotinib for RCC failed to demonstrate an advantage over available single-agent therapy in the second-line setting.

Pharmacokinetic modeling of doxorubicin pharmacokinetics in dogs deficient in ABCB1 drug transporters.

BACKGROUND: The identification of dogs defective in ATP-binding cassette transporter B1 (ABCB1, MDR1) activity has prompted questions regarding pharmacokinetics (PK), efficacy and toxicity of ABCB1 substrates in these dogs. HYPOTHESIS/OBJECTIVES: Dogs defective in ABCB1 activity (ABCB1(null)) have doxorubicin (DOX) PK different from that of normal dogs (ABCB1(wt)). Utilization of a physiologically based pharmacokinetic (PBPK) model allows computer simulation to study this polymorphism's impact on DOX PK. ANIMALS: None. METHODS: A virtual ABCB1(wt) dog population was generated and DOX distribution, elimination, and metabolism simulated by PBPK modeling. An in silico population of virtual dogs was generated by Monte Carlo simulation, with variability in physiologic and biochemical parameters consistent with the dog population. This population was used in the PBPK model. The ABCB1 components of the model were inactivated to generate an ABCB1(null) population and simulations repeated at multiple doses. Resulting DOX levels were used to generate PK parameters. RESULTS: DOX exposures in the ABCB1(null) population were increased in all simulated tissues including serum (24%) and gut (174%). Estimated dosages in the ABCB1(null) population to approximate exposure in the ABCB1(wt) population at a dose of 30 mg/m(2) were 24.8 +/- 3.5 mg/m(2) for serum and 10.7 +/- 5.9 mg/m(2) for gut. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest that serum DOX concentrations are not indicative of tissue exposure, especially those with appreciable ABCB1 activity, and that gastrointestinal (GI) toxicosis would be dose limiting in ABCB1(null) populations. Dosage reductions necessary to prevent GI toxicosis likely result in subtherapeutic concentrations, thereby reducing DOXs efficacy in ABCB1(null) dogs.

A biologically based model of growth and senescence of Syrian hamster embryo (SHE) cells after exposure to arsenic.

We modified the two-stage Moolgavkar-Venzon-Knudson (MVK) model for use with Syrian hamster embryo (SHE) cell neoplastic progression. Five phenotypic stages are proposed in this model: Normal cells can either become senescent or mutate into immortal cells followed by anchorage-independent growth and tumorigenic stages. The growth of normal SHE cells was controlled by their division, death, and senescence rates, and all senescent cells were converted from normal cells. In this report, we tested the modeling of cell kinetics of the first two phenotypic stages against experimental data evaluating the effects of arsenic on SHE cells. We assessed cell division and death rates using flow cytometry and correlated cell division rates to the degree of confluence of cell cultures. The mean cell death rate was approximately equal to 1% of the average division rate. Arsenic did not induce immortalization or further mutations of SHE cells at concentrations of 2 microM and below, and chromium (3.6 microM) and lead (100 microM) had similar negative results. However, the growth of SHE cells was inhibited by 5.4 microM arsenic after a 2-day exposure, with cells becoming senescent after only 16 population doublings. In contrast, normal cells and cells exposed to lower arsenic concentrations grew normally for at least 30 population doublings. The biologically based model successfully predicted the growth of normal and arsenic-treated cells, as well as the senescence rates. Mechanisms responsible for inducing cellular senescence in SHE cells exposed to arsenic may help explain the apparent inability of arsenic to induce neoplasia in experimental animals.

Alterations in P-glycoprotein expression in mouse tissues by doxorubicin: implications for pharmacokinetics in multiple dosing regimens.

The purpose of the studies presented here is to determine if alterations in doxorubicin (DOX) pharmacokinetics that seem to occur following multiple-dosing are due to changes in DOX elimination via P-glycoprotein (PGP) mediated transport in the liver, kidney and gut. A pharmacokinetic study in female Balb/c mice was carried out with blood and tissue DOX levels measured in animals following a single DOX treatment (6 mg/kg), and in animals following a second DOX treatment after receiving a DOX treatment a week earlier. The pharmacokinetics of DOX in blood and tissues was altered by earlier exposure to DOX, as the animals that were treated once a week for 2 weeks showed an increased rate of DOX elimination from blood and tissues following the second treatment. Immunoblot analysis of PGP expression in liver and kidney from naïve and DOX-treated mice showed an approximately 1.2-fold elevation of PGP protein in these tissues in response to DOX exposure. Immunohistochemical staining of liver and small intestine sections for PGP showed 1.6-fold and 1.9-fold increases, respectively, in the DOX-treated tissues. These results have implications both in multiple-dosing regimens, as well as multiple-drug regimens, where DOX is used in combination with other drugs that are substrates for PGP-mediated efflux. Increases in PGP expression in both hepatic and extrahepatic tissues can lead to changes in the pharmacokinetics of DOX, as well as other drugs that are transported by PGP.

Comparative toxicity of dithiocarbamates and butadiene metabolites in human lymphoid and bone marrow cells.

Apparent differences in the pattern of leukemia risk have been observed between workers employed in 1,3-butadiene (BD) monomer production and those working in styrene-butadiene rubber production (SBR). There are a number of possible explanations for these discrepancies, including differences in disease classification and diagnosis as well as possible quantitative and qualitative differences in occupational exposure between these two industries. This led us to evaluate the possibility that the pattern of disease observed in SBR might be influenced by the presence of an important class of biologically reactive chemicals, dithiocarbamates (DTC), that were present in SBR but not BD monomer production. Therefore, we compared the immunotoxic and hematotoxic activities of DTC and BD metabolites in human immune and hematopoietic cells. Relative to the mouse, human CD34+ bone marrow cells are relatively resistant to the direct effects of BD metabolites, with only the bis-oxide producing any evidence of suppression of clonogenic response at concentrations between 1 and 10 microM. Similarly, treatment of human CD4+ lymphocytes with known (2,3-epoxybutene) and putative BD metabolites (D,L-butane-bis-oxide, (2S,3R)-3-epoxybutane-1,2-diol) does not result in appreciable T-cell toxicity at concentrations likely to be encountered in vivo. In contrast, treatment of human cells with DTC at concentrations as low as 100 nM results in significant suppression of hematopoietic clonogenic response and T-lymphocyte function. Additional studies in our laboratory and others suggest a role for copper in DTC toxicity in both human lymphocytes and bone marrow cells, although the pattern of altered transcriptional regulation observed is markedly different in these two cell populations. These results are consistent with the pattern of DTC toxicity previously observed in clinical and molecular studies.

A physiologically based pharmacodynamic analysis of hepatic foci within a medium-term liver bioassay using pentachlorobenzene as a promoter and diethylnitrosamine as an initiator.

A stochastic clonal growth model for describing quantitative changes in size and number of putative preneoplastic lesions was modified to analyze the time-course information of cell proliferation and glutathione S-transferase pi (GST-P) foci within a medium-term bioassay. The study used F344 rats and a single initiating event using diethylnitrosamine (200 mg/kg ip) at Week 0. After a 2-week recovery period, chemical treatment began by gavage administration of pentachlorobenzene (PeCB; 100 micromol/kg/day, 7 days/week) in a corn oil vehicle and continued for 6 weeks. One week after beginning gavage dosing, a two-thirds partial hepatectomy was performed and the animals were serially euthanized at 48, 120, 168, 624, and 840 h postsurgery, which corresponds to 216, 288, 336, 792, and 1008 h following the beginning of PeCB treatment, respectively. For analysis, two types of models were evaluated for describing the time-course changes in GST-P foci. First, a sequential model describing the transformation of normal cells into a homogenous initiated cell population (i.e., one-cell model). Second, a two-cell model that describes a heterogeneous foci population by splitting the initiated cell population into two distinct types. In our study, the one-cell model was unable to adequately represent the time-course data for changes in both size and number of foci. In contrast, the two-cell model, which was parameterized to describe a negative selection mechanism, produced adequate simulations of both the size and number of foci. This model-based analysis suggested that the differences between PeCB-treated and untreated animals were primarily in parameters involving the rates of cell death.