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The emphasis on biomarkers as a new approach often has lead to expectations that the ideal biomarker must be novel and/or exotic. To answer the pertinent questions during drug development, teams often embark on the quest for the “unicorn” of biomarkers, sometimes resulting in the best and most practical test being overlooked as the search progresses for elusive methods. This article highlights examples of unicorn, horse, and mule biomarker approaches. Keywords: biomarkers; drug development; chronic myeloid leukemia (CML); pharmacogenetics; anticancer drug

Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, binds with high affinity to the α(2)δ subunit of voltage-gated calcium channels and exerts analgesic, anxiolytic, and antiseizure activities. Two-year carcinogenicity studies were completed in B6C3F1 and CD-1 mice and two separate studies in Wistar rats. Doses in mice were 200, 1000, and 5000 mg/kg/day, with systemic exposures (AUC(0-24 h)) up to 31 times the mean exposure in humans, given the maximum recommended clinical dose. In rats, doses were 50, 150, and 450 mg/kg/day in males and 100, 300, and 900 mg/kg/day in females; systemic exposures up to 24 times were achieved in clinical trials. In both strains of mice, pregabalin treatment was associated with an increased incidence of hemangiosarcoma primarily in liver, spleen, and bone marrow. The incidence of hemangiosarcoma was higher in B6C3F1 mice than in CD-1 mice, consistent with its spontaneous incidence. Pregabalin did not increase the incidence of any other tumor type in rats and was not genotoxic, based on an extensive battery of in vivo and in vitro tests in bacterial and mammalian systems. Thus, pregabalin is a single-species, single tumor-type, nongenotoxic mouse carcinogen. Hemangiosarcomas occurring in mice treated with pregabalin were genotypically distinct from hemangiosarcomas induced by genotoxic carcinogens in humans with respect to ras and p53 mutation patterns and were similar to spontaneous tumors. Furthermore, there was a strong association between pregabalin treatment and bone marrow changes in these studies in mice, suggesting a possible link between the effects observed in bone marrow and the increase in tumor incidence in pregabalin-treated mice.

The local lymph node assay (LLNA) is used to assess the skin sensitization potential of chemicals. In the standard assay, mice are treated topically on the dorsum of both ears with test substance for 3 days. Following 2 days of rest, the initiation of the hypersensitivity response is evaluated by injecting (3)H-thymidine into a tail vein, and then measuring the levels of radioisotope incorporated into the DNA of lymph node cells draining the ears. In the current study, BALB/c mice were treated with the contact sensitizers hexylcinnamic aldehyde (HCA) and oxazolone, and the nonsensitizer methyl salicylate. The proliferative response of lymph node cells was evaluated in an ex vivo assay, in which isolated cells were cultured in vitro with (3)H-thymidine. Treatment of mice with HCA at 5-50% resulted in concentration-related increases in (3)H-thymidine incorporation, with stimulation indices ranging from 3 to 14. Low animal-to-animal variability was seen in three replicate assays testing HCA at 25%. As anticipated, the proliferative response induced by the potent sensitizer oxazolone at 0.25% was greater than HCA at all concentrations tested. Stimulation indices of 1.5 and 3 were seen in two independent experiments with methyl salicylate. These equivocal findings were likely due to the irritancy properties of the compound. Importantly, measuring ex vivo (3)H-thymidine incorporation was more sensitive than evaluating lymph node weight and cellularity, and in vitro bromodeoxyuridine incorporation. Furthermore, the results of the ex vivo LLNA were comparable to the standard assay. This study provided evidence that supports the use of an ex vivo LLNA for hazard assessment of contact hypersensitivity.

Timing is Everything Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a robust platform for protein profiling and differential gene expression analysi...

Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to evaluation of much larger numbers of cells clearly demonstrate the usefulness of flow cytometry method in the routine micronucleus evaluation.

Platelet satellitosis of polymorphonuclear cells is a phenomenon induced or enhanced by the anticoagulant EDTA. In contrast with previously reported studies, the subject in the present case did not demonstrate platelet satellitism but was profoundly pseudothrombocytopenic owing to platelet phagocytosis. Virtually all polymorphonuclear leukocytes and monocytes contained numerous ingested platelets in contrast with previous cases in which phagocytosis was observed only rarely and involved ingestion of single cells. The phenomenon was documented by immunocytochemical staining and transmission electron microscopy. Autoantibodies were detected in EDTA-anticoagulated blood. However, neither platelet antibody nor phagocytosis was present when heparin, acid-citrate dextrose, or citrate was used as an alternative anticoagulant. The antibody was not temperature dependent. Mixing studies showed the transfer of the phagocytosis phenomenon to healthy donors. Although platelet function assays are typically normal in EDTA-dependent platelet satellitism, this subject showed no secondary aggregation wave in response to adenosine diphosphate and depressed adenosine triphosphate release with collagen, adenosine diphosphate, and arachidonic acid.

Hemangiosarcomasare uncommon malignant endothelial cell tumors in humans and experimental animal species. The mechanisms giving rise to these tumors are poorly understood even though the histotypes are comparable between humans and rodents. Activating mutations in cellular ras protooncogenes have been detected in sporadic and chemically induced human and rodent hemangiosarcomas. Ras activation significantly modulates tumor angiogenesis, suggesting that mutations in ras genes might be causally related to vascular tumorigenesis. To more clearly define the role of ras in experimental vascular tumorigenesis, mutations in the Ki- and Ha-ras genes were characterized in 63 hemangiosarcomas that arose unexpectedly in control and treated B6C3F1 mice during a two-year carcinogenicity study of the thiazolidinedione troglitazone. DNA was extracted from paraffin sections of mouse hemangiosarcomas, control liver, or positive control hepatocellular carcinomas with defined mutations in the Ki- or Ha-ras genes. Exons 1 and 2 of the Ki- and Ha-ras genes were independently amplified using primer extension preamplification/locus-specific heminested PCR, and PCR amplicons were directly sequenced to identify mutations in codons 12, 13, or 61. Activating mutations were detected in 3 of 63 hemangiosarcomas: a single G-->A transition in the second position of Ki-ras codon 13 in a tumor from a treated animal and two G-->T transversions in the second position of Ha-ras codon 13, one in a single tumor from a control animal and one in a tumor from a treated animal. These mutations are consistent with endogenous mutagenesis arising from oxidative DNA damage. The low frequency of mutation (

Inflammatory lung injury is probably regulated by the balance between proteases and protease inhibitors together with oxidants and antioxidants, and proinflammatory and anti-inflammatory cytokines. Rat tissue inhibitor of metalloprotease-2 (TIMP-2) and secreted leukoprotease inhibitor (SLPI) were cloned, expressed, and shown to be up-regulated at the levels of mRNA and protein during lung inflammation in rats induced by deposition of IgG immune complexes. Using immunoaffinity techniques, endogenous TIMP-2 in the inflamed lung was shown to exist as a complex with 72- and 92-kDa metalloproteinases (MMP-2 and MMP-9). In inflamed lung both TIMP-2 and SLPI appeared to exist as enzyme inhibitor complexes. Lung expression of both TIMP-2 and SLPI appeared to involve endothelial and epithelial cells as well as macrophages. To assess how these endogenous inhibitors might affect the lung inflammatory response, animals were treated with polyclonal rabbit Abs to rat TIMP-2 or SLPI. This intervention resulted in significant intensification of lung injury (as revealed by extravascular leak of albumin) and substantially increased neutrophil accumulation, as determined by cell content in bronchoalveolar lavage (BAL) fluids. These events were correlated with increased levels of C5a-related chemotactic activity in BAL fluids, while BAL levels of TNF-α and chemokines were not affected by treatment with anti-TIMP-2 or anti-SLPI. The data suggest that endogenous TIMP-2 and SLPI dynamically regulate the intensity of lung inflammatory injury, doing so at least in part by affecting the generation of the inflammatory mediator, C5a.

In this study, we determined in vivo morphologic effects of continuous intravenous infusion of recombinant human epidermal growth factor (EGF) in adult Wistar rats. The EGF used consisted of the amino acid residues 1-48 of the human 53-amino-acid EGF molecule, purified from transfected Escherichia coli. Doses of 25, 100, or 250 micrograms/kg body weight were administered using Harvard digital syringe infusion pumps for 4 weeks. At necropsy, the submandibular salivary glands, Harderian glands, liver, kidneys (females only), and ovaries were enlarged and urinary bladders were thickened in 100- and 250-micrograms/kg rats. Numerous tissues of the 100- and 250-micrograms/kg rats contained hyperplastic epithelial cells, and selected organs also had mesenchymal cell proliferation. Epithelial proliferation was most pronounced in the trachea, nasal cavity, nasolacrimal duct, tongue, stomach, small intestine, large intestine, urinary tract, salivary gland ducts, and Harderian gland. Periportal hepatocytes were hypertrophic, correlating with increased liver weight. In addition, mesenchymal cell proliferation was evident in the gastric mucosa lamina propria and in heart valves in 100- and 250-micrograms/kg rats. Increased ovarian weight correlated with increased number and size of corpora lutea and an increased incidence of luteal cysts. Continuous systemic exposure of adult Wistar rats to high doses of EGF resulted in generalized epithelial hyperplasia and tissue-selective mesenchymal proliferation.

CI-959 is an orally effective inhibitor of cellular activation in both in vitro and animal models. To assess the effects of CI-959 on immune function, male Fischer 344 rats were evaluated for splenic T- and B-lymphocyte populations, antibody-forming cell response to sheep red blood cells (sRBC), concanavalin A and pokeweed mitogen-induced lymphocyte proliferation, Natural Killer cell activity, and reticuloendothelial system clearance of sRBC. Host resistance was measured in female B6C3F1 mice using Listeria monocytogenes, Streptococcus pneumonia, and B16F10 melanoma models. CI-959 was administered to both species of rodents at 25, 50, and 75 mg/kg/day for 14 days. A vehicle control and two positive controls (cyclophosphamide and dexamethasone) were run concurrently. CI-959 generally did not suppress immunological responses in rats at doses lower than those which also altered body weight gain and reduced spleen and thymus weights. Natural Killer cell activity was significantly reduced at 50 and 75 mg/kg CI-959. At 75 mg/kg rats also exhibited a reduction in ability to make anti-sRBC antibody. The number of T- and B-lymphocytes, proliferative response to mitogens, and macrophage activity of the reticuloendothelial system were not affected by CI-959. CI-959 also did not alter resistance of mice to Listeria monocytogenes, Streptococcus pneumoniae, or B16F10 melanoma cells. Based on these ex vivo and in vivo assays, the rodent immune system does not appear to be a sensitive or toxicologically important target for CI-959.

Subacute Toxicity of a Purine Nucleoside Phosphorylase Inhibitor in Rats. Wolfgang, G. H. I., Bleavins, M. R., Hallak, H., Kasali, O. B., and Urda, E. (1995). Fundam. Appl. Toxicol. 28, 51-58. Rats received daily oral doses of 15, 50, 150, or 200 mg/kg CI-1000 for 4 weeks. Doses were selected based on findings from a 2-week range-finding study where doses of 250 and 500 mg/kg resulted in mortality. In the 4-week study, females given 200 mg/ kg were sacrificed during Week 2 due to poor condition. Serum creatinine and urea nitrogen increased 2- to 2.5-fold in females given 200 mg/kg. Dose-related increases in urine volume, urinary protein excretion, and osmolar excretion occurred in both sexes beginning at 50 mg/kg. Kidney weights increased 9-40% in both sexes at ⩾50 mg/kg; histopathologic changes were confined to the 150 and 200 mg/kg groups. At Week 4, T-suppressor/cytotoxic lymphocytes were reduced 43% and T-helper/inducer lymphocytes were reduced 22% in males given 200 mg/kg. In females, T-sup-pressor/cytotoxic lymphocytes were significantly decreased (approximately 40%) at 50 and 150 mg/kg, with no significant effects on T-helper/inducer lymphocyte populations. At Week 8, following 4 weeks without treatment, T-lymphocyte subpopulations were similar in control and drug-treated groups. B-lymphocyte counts and percentages were increased at Weeks 4 and 8 in males receiving 150 or 900 mg/kg. Thymic weights decreased at Week 4 at doses of 150 and 200 mg/kg. Plasma CI-1000 levels were higher in females than in males at all doses except 15 mg/kg; Cmax and AUC values were largely dose proportional in both sexes. In summary, CI-1000 was well-tolerated at doses of 15, 50, and 150 mg/ kg with no adverse effects occurring at 15 mg/kg. Drug-induced changes in the kidney were mild and reversible. Immunomodulatory effects were noted at doses of 50 mg/kg or higher.

Characterization of immune cell subpopulations in the cynomolgus monkey was performed using a direct immunofluorescence technique adaptable for routine and repeated monitoring. This whole blood procedure is faster and requires less volume than conventional density gradient isolation methods. Low intra- and inter-animal variations were seen in hematology parameters and in CD4, CD8, and CD20 lymphocyte subtypes. CD4 values were 28% of lymphocytes in males and 30% in females. Fifty-six percent were CD8+ in males and 54% in females. CD4:CD8 ratios were approximately 0.5 in both sexes. This proportion is the reverse of that observed in humans, but appears normal for the cynomolgus. Consistent with values reported for humans, approximately 12% of cynomolgus peripheral blood lymphocytes were CD20+. Greater than 95% of the lymphocytes present in blood were identified as CD4, CD8, or CD20 positive.

The emphasis on biomarkers as a new approach often has lead to expectations that the ideal biomarker must be novel and/or exotic. To answer the pertinent questions during drug development, teams often embark on the quest for the “unicorn” of biomarkers, sometimes resulting in the best and most practical test being overlooked as the search progresses for elusive methods. This article highlights examples of unicorn, horse, and mule biomarker approaches. Keywords: biomarkers; drug development; chronic myeloid leukemia (CML); pharmacogenetics; anticancer drug

Alpha 2u-Globulin (alpha 2u) nephropathy is a male rat-specific condition caused by a diverse group of xenobiotics. Features of this nephropathy include hyaline droplet accumulation in proximal tubules, tubular epithelial necrosis and regeneration, exacerbation of spontaneous renal disease, and induction of renal epithelial tumors. Nephrocarcinogenicity of compounds that cause this nephropathy may be a consequence of increased proximal tubular proliferation resulting from cell injury. These studies document alpha 2u nephropathy without primary renal epithelial tumors in male Wistar rats administered 1-(aminomethyl)cyclohexaneacetic acid (gabapentin), a therapeutic agent with antiepileptic/anticonvulsant properties. In a series of preclinical studies gabapentin was administered to rats at the following doses and durations: 50 and 2000 mg/kg for 2 weeks; 250, 1000, 2000, and 3000 mg/kg for 13 weeks; 250, 1000, and 2000 mg/kg for 52 and 104 weeks. Renal effects were evaluated by biochemical, immunocytochemical, histopathologic, and ultrastructural techniques. Reversible increases in size and distribution of hyaline droplets within proximal tubular epithelium occurred through 1 year of treatment at a severity that was dose-dependent. In males given 2000 mg/kg, alpha 2u accumulation, degeneration, and necrosis of the P2 segment and intraluminal cellular casts were seen after 2 days of treatment. In the 2-week study, the size and number of phagolysosomes containing alpha 2u and the renal tissue alpha 2u increased with increasing dose and time. By Day 7, polymorphic crystalline inclusions were abundant in phagolysosomes of 2000 mg/kg males. In subchronic and chronic studies, spontaneous glomerulonephrosis was exacerbated in males given 2000 mg/kg, and, interestingly, no drug-related effect on renal tumor incidence was observed. To the best of our knowledge, this is the first documentation of the absence of nephrocarcinogenic effect in male rats treated for up to 104 weeks with a compound that causes acute and chronic lesions of alpha 2u nephropathy.

The effect of CI-949, a novel inhibitor of allergic mediator release, on immune function was assessed with holistic mouse models of immunocompetence. Resistance to the bacterial pathogens Listeria monocytogenes and Streptococcus pneumoniae and the B16F10 melanoma cell line was used to evaluate the potential of CI-949 to affect immune function. CI-949 treatment of female B6C3F1 mice increased pulmonary tumor burden at 100 mg/kg/day in the B16F10 melanoma model, with a no effect level of at least 50 mg/kg/day. A correlation was seen between decreased clearance of the B16F10 cells and increased tumor burden. However, CI-949 produced this effect only at the maximum tolerated dose. No effect of the drug was seen in the S. pneumoniae model. Host resistance to L. monocytogenes was increased after CI-949 administration, with the no adverse effect level in this model being at least equivalent to the top dose of 100 mg/kg/day. Therefore, the immune system does not appear to be adversely affected or to be a specific target for CI-949 even at an overtly toxic dose.

Ozone is a photochemical oxidant which reacts with a variety of biological molecules. In experimental animals the toxicity of ozone results from damage to cells in the lung and is associated with the process of reactive repair and an influx of inflammatory cells. In this work we used an indirect immunofluorescence technique to study the effect of ozone on T and B lymphocytes with special reference to their presence in ozone-induced pulmonary lesions. BALB/c mice were exposed to ozone at a concentration of 0.7 ppm for 20 hr per day, 7 days per week, for 4 or 14 days. Control mice were housed in comparable chambers lacking ozone. Frozen sections of lung were prepared and stained for the surface markers Thy-1.2 (T lymphocyte) and sIgM (B lymphocyte). These experiments showed that T lymphocytes infiltrate the lung lesions during ozone inhalation, and increase their presence as ozone exposure continues. They were present in the centroacinar region and tended to occur in clusters within the ozone-induced lesions. In contrast, the infiltration of B lymphocytes was virtually nonexistent with few or no sIgM positive cells present in the lesions after either 4 or 14 days of exposure to ozone. These results show that T lymphocytes occur within the sites of ozone-induced damage and support that this cell type plays a role in the reactive host response to ozone inhalation.

Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also because antigen-antibody interactions are concentration-dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody-based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted, and the other color represents the amount of the antigen that is used to quantify the level of protein expression. When compared with median fluorescence intensity alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins interleukin (IL)-1beta, IL-5, IL-6, and macrophage inflammatory proteins 1alpha and 1beta. The levels of protein expression seen with the antibody microarray was compared with that obtained with Western blot analysis, and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as Western blot analysis, and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood.

The microsomal enzyme cytochrome P450 2D6 (CYP2D6), also known as debrisoquine 4-hydroxylase, is involved in the oxidative metabolism of many widely used drugs, including neuroleptics, tricyclic antidepressants, antiarrhythmics, and β-adrenergic blocking agents (1). Polymorphisms of CYP2D6 are the best characterized examples of genetically mediated effects on a drug-metabolizing enzyme of clinical importance (2). When a drug that is a CYP2D6 substrate is taken by different individuals, it is not uncommon to observe large differences in plasma concentrations at steady state. This is explained, in part, by the three clinically distinct phenotypes associated with the CYP2D6 gene, normal metabolizers, poor metabolizers, and rapid metabolizers. In normal metabolizers, steady-state plasma drug concentrations fall within the desired therapeutic range and toxic effects are nonexistent or minimal. In fast-metabolizer individuals, desired concentrations are below therapeutic, and these patients generally do not respond at the recommended dosing regimen. In poor-metabolizer individuals, drug concentrations are above therapeutic level and undesired toxicity can be evoked.

High-density microarrays are useful tools to study gene expression for the purpose of characterizing functional tissue changes in response to the action of drugs and chemicals. To test whether high-density expression data can identify mechanisms of toxicity and to identify an unknown sample through its RNA expression pattern, groups of male Wistar rats were administered 6 hepatotoxicants. The compounds chosen for this study were microcystin-LR (MLR), phenobarbital (PB), lipopolysaccharide (LPS), carbon tetrachloride (CT), thioacetamide (THA), and cyproterone acetate (CPA). These hepatotoxicants are known to induce adverse liver effects through different mechanisms. Liver mRNA was isolated and used to generate biotinylated cRNA for hybridization to a custom 1,600–rat gene DNA microarray. Treatment correlation matrices analyzed hybridization data from a hepatotoxicant-blinded sample, with gene expression coefficients (GEC) evaluated by means of hierarchical cluster analysis and visual representation as dendrograms. The experimental liver toxicity from the different treatments was confirmed by means of concurrent histopathology, liver enzymes, and bilirubin assays. This toxico genomic analysis identified multiple genes and groups of genes that were affected by the hepatotoxicants on study, indicating that high-density microarray expression data are useful to identify groups of genes involved in toxicity. In addition, the mRNA expression profile of an unidentified sample can be accurately identified when compared with the expression profiles resident in the data set. This study supports the use of gene expression–profiling technology to determine or to predict toxic liver effects. (HEPATOLOGY 2001;33:1239-1258.)

AIMS: The two electron reduction of quinones to hydroquinones by NAD(P)H quinone oxidoreductase (NQO1) plays an important role in both activation and detoxification of quinone and similarly reactive compounds. A single nucleotide polymorphism at exon 6 leads to an amino acid change at codon 187 from proline to serine. The variant allele has been associated with decreased NQO1 enzyme activity and increased cancer risks. The aim of this study was to develop a rapid genotyping procedure for epidemiological and clinical research into the potential biological and toxicological implications associated with this genetic polymorphism. METHODS: A high throughput genotyping method using fluorogenic probes has been developed to screen this single nucleotide polymorphism. This assay utilises the 5' nuclease activity of Taq polymerase in conjunction with fluorogenic TaqMan probes. The TaqMan genotyping procedure was validated by a restriction fragment length polymorphism method and direct sequencing. RESULTS: This method can be used for the rapid screening of known polymorphisms in large populations. In a population of 143 unrelated individuals, Pro/Pro (wildtype), Pro/Ser (heterozygous), and Ser/Ser (mutant) genotypes were 69.2%, 26.6%, and 4.2%, respectively. CONCLUSIONS: This genotyping method is highly accurate and could be applied to automated large scale genotyping studies.

Hemangiomas and hemangiosarcomas are uncommon in rodents and humans and, as such, the mechanisms giving rise to these tumors are poorly understood. Inactivating mutations in the p53 gene have been detected in sporadic and chemically induced human and rodent hemangiosarcomas. Additionally, experimental ablation of p53 function in mice by targeted gene disruption increases the incidence of both spontaneous and carcinogen-induced vascular tumors. These findings implicate p53 disruption in vascular tumor development. In this study, we characterized p53 inactivation immunocytochemically and by gene sequencing in a large number of vascular tumors that developed in B6C3F1 mice during a long-term (2-year) study of the thiazolidinedione troglitazone. For comparative purposes, a murine hemangiosarcoma induced by polyoma middle-T antigen, which transforms endothelial cells via a p53-independent mechanism, five spontaneous human hemangiosarcoma specimens, and species-specific positive control tissues were also evaluated by immunocytochemistry for p53 inactivation. While 20% of the human hemangiosarcomas and all positive control tissues expressed significant levels of nuclear p53, indicating functional inactivation of the protein, none of the 161 mouse vascular tumors studied expressed detectable p53 protein. The absence of inactivating mutations was confirmed in eight of the histologically most malignant mouse hemangiosarcomas by sequencing exons 5 to 8 of the p53 gene. These results demonstrate that p53 inactivation did not play a role in development of the vascular tumors seen in the long-term study of troglitazone, and they indicate that loss of p53 function is not essential for vascular tumor development in mice.

A delayed-type hypersensitivity (DTH) test commonly used for humans was adapted for use with cynomolgus monkeys (Macaca fascicularis). Pilot experiments showed naive animals had poor response rates and inconsistent reactivity to the antigens. In an exploratory phase, it was determined that monkeys could be experimentally sensitized by immunization with commercially available antigens. Animals were then sensitized with various concentrations of diphtheria and tetanus toxoids, Candida, and Trichophyton in the dose-response phase. Antigens were injected intradermally (i.d.) 3 times over a 7-day period and monkeys were tested 14 days after the last injection. Responses were measured 24, 48, and 72 h post-challenge, with skin biopsies taken from two animals per group at the 24 h interval. Optimal concentrations were 1.2 Lf diphtheria, 6 Lf tetanus, 1000 PNU Candida, and 1000 PNU Trichophyton. These concentrations produced the best balance between DTH responses, homogeneity of dermal mononuclear cell infiltrate and lowest frequency of undesirable skin reactions. Positive responses were seen at 24 and 48 h post-challenge and were waning by 72 h. DTH responses were inhibited by topical corticosteroids. The final phase of these studies assessed whether sensitization of naive animals could be achieved using subcutaneous (s.c.) administration of the optimal antigen concentrations. Comparable responses to i.d. sensitization were obtained and skin sores did not develop at injection sites. These studies show that the DTH test adapted to monkeys was reproducible, minimally invasive, did not require sacrifice of the test animal, allowed repeated measurements and paralleled the reactions observed in humans.

CI-1000, a 9-deazaguanine analog, is an orally active and reversible inhibitor of purine nucleoside Phosphorylase (PNP). In vitro, CI-1000 inhibits human T-lymphoblast replication and the mixed lymphocyte response, as well as facilitating the intracellular accumulation of deoxyguanosine triphosphate (Gilbertsen et al., 1992; Dong and Gilbertsen, 1993; Wilburn et al. 1993). These changes are consistent with inhibition of PNP (Osborne and Scott, 1983; Fairbanks et al., 1990; Gilbertsen and Sircar, 1990) and the drug is approximately 10-fold more potent than a structurally related compound (Gilbertsen et al., 1991a, 1991b, and 1992).

Feeding adult mink and European ferrets diets that contained 1, 5, or 25 mg/kg added hexachlorobenzene (HCB) resulted in reduced reproductive performance as indicated by decreased litter size, increased percentage of stillbirths, increased kit mortality, and decreased early kit growth. Diets treated with 125 or 625 mg/kg HCB were lethal to the adults of both species. In general, the mink were more sensitive to the toxic effects of HCB than were ferrets.

Adult female ferrets were found to absorb 85.4% of a single dietary dose of the polychlorinated biphenyl (PCB) mixture Aroclor 1254. Excretion in the days immediately following dosing yielded the greatest quantity of PCBs eliminated in a given time period. In general, urinary excretion represented one-tenth or less of the PCBs excreted via the feces. Placental transfer to the ferret kits was 0.01% (per kit) of the female's absorbed dose when exposure occurred during the first trimester of pregnancy and 0.04% (per kit) when the PCBs were administered during the third trimester. Transplacental exposure to PCBs was considerably less than that reaching the kits through the dam's milk. The ratio of placental to mammary transfer, following 1 week of lactation, was calculated to be approximately 1:15 for the offspring of first-trimester-dosed females and 1:7 for the offspring produced by females treated during the third trimester.

Adult female standard dark mink were exposed to hexachlorobenzene (HCB) at concentrations of 0, 1, and 5 ppm in the feed and bred with males on the same treatments. Female offspring were allowed to mature to 16–17 weeks and killed. At 16–17 weeks of age, HCB had no effect on body weights or liver weights. Hepatic cytochrome P-450 and ethoxyresorufin-O-deethylase were significantly increased 2.0- and 1.5-fold, respectively, in the 5-ppm treatment group. Electron microscopy failed to reveal proliferation of the smooth endoplasmic reticulum. No hepatic damage was observed. No changes in in vitro renal function, measured as accumulation of para-aminohippurate and tetraethylammonium by renal cortical slices, were detected in any treatment group. Histological examination of renal slices did not reveal any alterations in morphology. Fat was the predominate site of HCB disposition; samples from the 5-ppm treatment group contained 626.10 ± 12.01 ng HCB/g tissue. Whereas perinatal HCB administration has profound effects on the survival of off-spring born to exposed mink, only induction of hepatic mixed-function oxidases was observed in the surviving kits without any observable frank hepatotoxicity.

Hexachlorobenzene (HCB) was found to depress the lymphocyte blastogenic response of mink (Mustela vison) and European ferrets (Mustek putorius furo) to the plant lectin Concanavalin A (Con-A). A significant reduction in the incorporation of [3H]thymidine was seen in the lymphocytes from adult animals consuming 25 ppm dietary HCB for 8 months. This effect was observed in animals showing no physical signs of distress or toxicity. The in utero and early postnatal exposure of young mink to HCB also resulted in a significant depression of lymphocyte responsiveness. Exposure levels as low as 1 ppm in the dam's diet caused an impaired T-cell response. In the young ferrets exposed to HCB in utero and during nursing, no change in the stimulation index was seen. The monitoring of immunocompetence in animals exposed to toxicants, either through the diet or indirectly via their dam, provides a sensitive indicator of toxicity. Effects were seen in activation of lymphocytes from the mink and ferrets used in this study without grossly observable clinical signs being present.

A comparison was made of the induction responses of two PCBs (Aroclors 1016 and 1242) on the hepatic microsomal mixed function oxidase (MFO) system in two genetically similar species, mink and European ferrets, previously shown to differ significantly in sensitivity to these same Aroclors. Feeding Aroclors for 28 days at a subtoxic level (20 ppm) resulted in weak induction of the MFO system. Aroclor 1242 was a more potent inducer than Aroclor 1016. Using a dosing regime which resulted in toxic effects in mink but not in ferrets, greater induction was noted in ferrets compared to mink. Furthermore, only 3-methyl-cholanthrene (MC)-type induction was observed. Phenobarbital (PB) inducible enzymes were either not affected or were inhibited by Aroclor treatment. The results suggest an inverse relationship between extent of MC-type induction and toxicity in these species.

The effects of 3,3′,4,4′-tetrachloroazoxy-benzene (TCAOB) on the immune system of mice was examined and compared with cyclophosphamide (CY). This chemical can be produced as a result of microbial degradation of commonly used chloroaniline herbicides. Herbicides of the acylan-iline class generally have a low mammalian toxicity while remaining relatively inexpensive. Therefore, TCAOB could be formed anywhere in the environment where choroaniline pesticides are used. TCAOB treatment caused thymic atrophy and a decrease in white blood cell (WBC) count after sheep red blood cell (SRBC) immunization. A decline in the number of Lyt-1+ cells (T-helper lymphocytes) was seen in all TCAOB animals, with unimmunized mice also showing a decreased number of Lyt-2.2+ cells (T-suppressor lymphocytes). No change was found in the ratio of these two cell types. TCAOB did, however, result in a severe reduction in the number of plaque-forming-cells (PFCs) and in serum antibody concentration. CY caused a decrease in thymus weight, WBC count, the number of cells recovered per spleen, and the relative percentages of Lyt-l+ and Lyt-2.2+ cells recovered. The mice exhibited a lower lymphocyte blastogenic response to lipopolysaccharide (LPS) than control animals, but no change in Concanavalin A (Con-A) responsiveness. CY also resulted in a severe drop in the number of PFCs and the quantity of antibody produced following SRBC immunization.

Dietary exposure to 1000 ppm of supplemental Zn did not result in grossly observable Zn toxicity or Zn-induced Cu deficiency in adult mink. These same concentrations did, however, produce achromatrichia, alopecia, lymphopenia and a reduced rate of growth in the offspring produced by the Zn-treated females. These mink kits also exhibited profound immunosuppression. The in vitro blastogenic response of peripheral blood lymphocytes to concanavalin A was significantly (P less than 0.001) lower in kits born to Zn-treated dams than the response of those born to control dams. The depressed immunoresponsiveness was not a permanent defect since a normal lymphocyte response was seen approximately 14 weeks after weaning and being placed on an unsupplemented basal diet. The impaired lymphocyte reactivity is believed to be the result of altered DNA synthesis in these cells and/or an inhibition of macrophage functions necessary for normal response to the mitogen concanavalin A.

A search for the cause of reproductive complications and excessive newborn mortality in mink fed Great Lakes fish in the late 1960's led to the demonstrated toxicity of polychlorinated biphenyls (PCBs) in this carnivore. Studies were undertaken to quantitate the toxicity of several PCBs on mink and ferrets, to contrast placental transfer to the fetus and milk biotransfer to the newborn, and to compare PCB to polybrominated biphenyl (PBB) toxicity. Dietary levels as low as 2 ppm of Aroclor® 1254 impaired mink reproduction. Complete fetotoxicity for Aroclors 1242 or 1254 was determined to be less than 5 ppm. The dietary concentration lethal to 50 percent of the adult mink was calculated as 8.6 and 6.65 ppm for Aroclors 1242 and 1254, respectively. The ferret was found to be somewhat less sensitive to several of these chlorinated hydrocarbon compounds. PCB transfer to the newborn via milk was greater than placental transfer. PBB was not as fetotoxic as PCBs but was lethal to the adult at a lower dietary concentration.

Diets that contained various levels of supplemental Aroclor 1242 or Aroclor 1016 were fed to mink and ferrets to investigate the chronic toxicity of these PCBs in two closely related species. In mink, Aroclor 1242 was found to be more toxic than comparable or higher levels of Aroclor 1016. The Aroclor 1242 diets caused complete reproductive failure at levels as low as five ppm of the diet. Aroclor 1016 impaired reproduction less than Aroclor 1242. Although fewer females whelped and the four-week kit weights were less than the control animals, no outward signs of abnormalities beyond their smaller size were found in the kits whelped and nursed by dams fed Aroclor 1016. Ferrets were more resistant to the effects of either PCB mixture than were the mink, as noted by the lower mortality rate on the Aroclor 1242 diet and the almost normal level of reproduction on the Aroclor 1016 diet. Feeding Aroclor 1242 at 20 ppm resulted in complete reproductive failure, but was not fatal to adult ferrets. This finding is in sharp contrast to the 100% mortality of adult mink fed the same level. Although the chlorine content is similar in both compounds, Aroclor 1242 has a higher percentage of molecules with five or more chlorines per biphenyl. This difference in higher substituted biphenyl isomer content and/or the reduced levels of contaminants in the Aroclor 1016 mixture may be of major importance in evaluating the toxicity of these compounds.

Natural dark mink kit were fed a diet supplemented with 0, 25, 50, 100 or 200 ppm Cu from CuSO4 . 5H2O for 153 or 357 d. The shorter term Cu supplementation had no significant beneficial or adverse effects on mink body weight gains or hemoglobin or hematocrit concentrations, although plasma Cu concentrations were slightly elevated in the mink fed added Cu. Liver Cu concentrations were significantly increased only in the mink fed 200 ppm Cu. Liver Zn and Fe concentrations were not affected by the added Cu. Darker fur was observed in pelted males fed the higher levels of Cu. The reproductive performance of mink on the longer term Cu supplementation was not adversely affected, although greater kit mortality and reduced "litter mass" were a result of the higher Cu concentrations. The acute (21-d) ip LD50 concentrations of Cu sulfate and Cu acetate in adult mink were 7.5 and 5.0 mg/kg, respectively.

Polychlorinated biphenyls (PCBs) have become one of the most widespread and persistent environmental pollutants in the world’s terrestrial and aquatic ecosystems (Risebrough et al. 1968, Johnson and Manske 1976, Kalmaz and Kalmaz 1979). The desirable properties of these aromatic hydrocarbons (relative inertness to chemical and biological degradation, high flash points, and lipid solubility) fostered their use in a wide range of products and electrical systems (Bailey et al. 1970, Rhee and Plapp 1973). These same properties underlie PCB persistence in the environment, uptake by plants (Buckley 1982), and bioaccumulation and biomagnification in animal species (Kimbrough 1974, Kalmaz and Kalmaz 1979). The assessment of the hazard posed by PCBs to human beings and other animals has been confounded by the complexity of the commercial PCB mixtures. These problems arise both from the large number of possible isomers (Dustman et al. 1971) and the presence of contaminants such as chlorinated dibenzofurans (Bowes et al. 1975), chlorinated naphthalenes (Hutzinger et al. 1974), and chlorinated dibenzodioxins (Porter and Burke 1971). Kimbrough (1974) has published an excellent review of the toxicity and physical properties of PCBs to which the reader is reffered for further information.

Female European ferrets (Mustela putorius furo) absorbed 98.5% of a single dietary exposure of hexachlorobenzene (HCB). The HCB was found to readily cross the placenta and to be excreted in the milk of pregnant/lactating ferrets. After consuming HCB-treated feed, ferrets raising offspring excreted 50% of the initial dose by 32 d, while unbred ferrets achieved this same degree of HCB elimination in 41 d. The percentages of HCB excreted via the urine and feces were approximately 5 and 45%, respectively, in both groups at the 50% stage of elimination. Adipose tissue was the most significant long-term repository for HCB in the ferret. The other tissues analyzed for [14C]HCB showed a general relationship of increased radioactivity with increased fat content of the tissue. The ferrets with nursing kits were able to significantly reduce their body burden of HCB when compared to unbred females. The developing ferret kits were subjected to HCB insult both in utero and via dam's milk. The ratio of milk to placental exposure in the growing offspring was calculated to be 31:1. Thus, in addition to any toxic effects HCB may have on the adult reproducing population, the placental and mammary transfer of HCB constitutes a potential threat to the developing and growing animal.

After a single dose of [14C]hexachlorobenzene (HCB) via gavage into the crop, the accumulation of [14C]HCB in female bobwhite (Colinus virginianus) tissues occurred to the greatest extent in adipose tissue followed by skin, liver, brain, heart and kidney, whole blood, and muscle. There was a general relation between increasing HCB concentration and increasing fat content of the tissue. Absorption of [14C]-HCB was rapid with peak radioactivity occurring at 4 h in all tissues except for fat and skin, where it continued to rise until 32 and 16 h after dosing, respectively. Elimination of HCB from tissue was biphasic with phase I representing the combination of HCB excretion and HCB redistribution from tissue into fat stores. Phase II represented solely HCB excretion, which appeared to be a first-order process. The half-life of [14C]HCB in tissues, feces, and eggs ranged from 9-13 d regardless of HCB concentration. Radioactive HCB accumulation in egg yolk was a significant mechanism for the removal of this chemical from bobwhite and accounted for 50% of the total HCB excreted.

European ferrets fed a diet that contained 20 ppm Aroclor 1242 for several months developed elongated, thickened, and deformed toenails. The excessive nail growth was more conspicuous in the males than in the females and was especially pronounced in the hind feet. Histopathologic examination of the affected ferret toes revealed a hyperkeratosis at the junction of the skin and eponychium, and dysplasia at the root of the nail and matrix. Accumulations of keratinized debris, as well as nuclear and cytoplasmic degeneration of many cells were noted and acanthosis and parakeratosis were present. It is speculated that the abnormal toenail development may be attributed to certain contaminants in Aroclor 1242.

In the initial trial, adult mink and ferrets were administered hexachlorobenzene (HCB) via the feed at concentrations of 1, 5, or 25 ppm for 47 wk. Animals receiving 125 and 625 ppm HCB in the diet died before termination of the experiment, with female ferrets at the 125 ppm level displaying abnormal aggressiveness and hyperexcitability just prior to death. Hypothalamic serotonin (5-HT) was significantly elevated at all dose levels in mink, and cerebellar 5-HT was significantly elevated at 1 ppm in the ferret. Regional brain biogenic amine concentrations were also determined in the offspring of the female mink that were administered 1 and 5 ppm HCB. Hypothalamic dopamine (DA) concentrations were significantly depressed by 1 and 5 ppm in these kits. In a second study, adult male and female ferrets were administered 250 or 500 ppm HCB via the diet for 7 wk. Two animals at the 250-ppm level and 3 animals at the 500-ppm level died before termination of the experiment without showing behavioral changes. Of the remaining animals, 3 ferrets at 250 ppm and 1 ferret at 500 ppm showed slight aggressiveness and hyperexcitability during the last week of the experiment. Concentrations of 5-HT were significantly elevated at 500 ppm in the cerebral hemispheres and at 250 ppm in the midbrain of male ferrets, while in the females, 5-HT was elevated in the cerebral hemispheres at 250 ppm and in the hypothalamus at both 250 and 500 ppm. Norepinephrine (NE) concentrations were significantly elevated in the cerebellum of males exposed to 250 and 500 ppm, as were NE concentrations in the midbrain. HCB at 500 ppm caused a significant increase in medullary NE, while 250 ppm caused an increase in hypothalamic NE in males. The only change in regional brain dopamine (DA) concentrations occurred at 500 ppm HCB in the midbrain of males, where there was a significant elevation of this neurotransmitter.