Immunotoxic effects of cyclophosphamide and cyclosporine in the dog.

Limited non-clinical immunotoxicity data are available in the dog, although this is a major non-rodent species in regulatory safety studies. The present study aimed to test whether widely accepted immunotoxicity endpoints including lymphocyte subset immunophenotyping, the anti-KLH TDAR assay, and histological examination of the main lymphoid organs were reliable to detect immunosuppression induced by cyclosporine and cyclophosphamide in dogs and could, therefore, be used for non-clinical immunotoxicity evaluation in this species. Male and female Beagle dogs were treated orally from Day 1 for 4 weeks with 25 mg/kg cyclosporine daily, or with 2 mg/kg cyclophosphamide on 4 consecutive days each week, or the same volume of drinking water daily. Blood samples were withdrawn pre-test and on Days 11, 18, and 23 to measure standard hematology parameters and analyze lymphocyte subsets. All animals received an intramuscular injection of 5 mg KLH on Day 11. Sandwich ELISA assays were used to quantify anti-KLH IgM and anti-KLH IgG levels in blood samples taken pre-test, on Days 18 and 23, and pre-test, on Days 23 and 28, respectively. At the end of the treatment period, all animals were submitted to histological examination of lymphoid organs, liver, and kidneys. No signs of marked toxicity were observed. No changes in lymphocyte subsets, but markedly decreased primary anti-KLH IgM and IgG responses, and a slightly-to-markedly increased cortex/medulla ratio in the thymus were observed in cyclosporine-treated dogs. Lower total WBC counts correlating with lower total and B-lymphocyte subset and decreased germinal center development in mesenteric lymph nodes, but no changes in primary anti-KLH IgM and IgG responses were observed in cyclophosphamide-treated dogs. These results demonstrate that widely accepted immunotoxicity endpoints can adequately detect the effects of known immunosuppressive drugs in the dog and support the conclusion that it is a relevant animal species for immunotoxicity evaluation.

Development of a Delayed-Type Hypersensitivity (DTH) Model in the Cynomolgus Monkey.

Although a T-dependent antibody response (TDAR) assay is generally recommended as the first-line immune function assay in nonclinical immunotoxicity evaluation, second-line assays such as delayed-type hypersensitivity (DTH) to measure cell-mediated responses can provide helpful additional information. In this study, male Cynomolgus monkeys were injected intramuscularly either once or twice with 1 mg Keyhole Limpet Hemocyanin (KLH) or twice with a commercially available tetanus vaccine (40 IU tetanus toxoid + 0.06 mg aluminum hydroxide). All animals were subsequently challenged by intradermal injections of the same antigen or aluminum hydroxide after 4, 6 and 8 weeks. Clinical reactions at the injection sites were scored 24, 48 and 72 h post challenge. Skin biopsies were taken on completion of the observation period after each challenge for standard histological examination and immunolabeling using CD3 (T lymphocytes), CD19 (B lymphocytes) and CD68 (macrophages) antibodies. Tetanus toxoid induced stronger clinical reactions than KLH, whereas aluminum hydroxide induced no clinical reaction. Perivascular mononuclear cell infiltrates, a histopathological finding consistent with a DTH reaction, were seen after all challenges with tetanus toxoid or KLH, but not with aluminum hydroxide. Immunohistochemistry evidenced the presence of T lymphocytes and macrophages within these infiltrates. These results suggest that tetanus toxoid adjuvanted with aluminum hydroxide can induce a consistent DTH response for use as a model of cell-mediated response in Cynomolgus monkeys.

Recording of the full-field electroretinogram in minipigs.

PURPOSE: To test a simple electroretinographic protocol on a representative sample of minipigs. ANIMAL STUDIED: Minipig. PROCEDURES: Electroretinogram recordings were conducted on 162 healthy minipigs (81 males and 81 females) aged 4-6 months. After a 1.5-h light-adaptation period, the animals were anesthetized with general anesthesia. First, binocular full-field photopic electroretinogram recordings were conducted under photopic conditions. Subsequently, scotopic electroretinogram recordings were conducted during dark-adaptation periods every 4 min for a 20-min period. At the end of this period, the maximal combined rod-cone response was recorded by measuring the retinal response to a single high-intensity flash. We used sclerocorneal clip electrodes as active electrodes and needle electrodes as reference and ground electrodes. RESULTS: The a-wave and b-wave peak times and amplitudes have been measured and statistically analyzed. For each of the statistical comparisons, normality and homogeneity of variances were evaluated. No significant gender differences were observed, with the exception of a higher b-wave amplitude for the photopic ERG recordings observed in females when compared to males (48.14 ± 12.909 µV vs. 42.88 ± 10.666 µV; P = 0.005). The process of dark adaptation was evaluated, and the maximal combined rod-cone response was measured (a- and b-waves amplitude and peak time). CONCLUSIONS: We conducted photopic and scotopic electroretinogram recordings from a protocol based on light adaptation followed by dark adaptation using sclerocorneal clip electrodes, which allows quick assembly and examination.

Systemic exposure to parabens: pharmacokinetics, tissue distribution, excretion balance and plasma metabolites of [14C]-methyl-, propyl- and butylparaben in rats after oral, topical or subcutaneous administration.

Parabens (PB) are preservatives used in food, drugs and personal care products preventing microbial and fungal contamination. We investigated ADME profiles of [14C]-methyl-, propyl- or butylparaben (MP, PP, BP) following single oral, dermal or subcutaneous (BP) doses at 100 mg/kg to Sprague-Dawley rats. Plasma Cmax and AUC values after oral or subcutaneous doses were 4- to 10-fold higher relative to respective values after dermal administration. tmax ranged from 0.5, 2 or 8 h after oral, subcutaneous or dermal administration, respectively. MP produced higher blood Cmax and AUC levels relative to those after PP or BP. Following oral or subcutaneous administration, urinary excretion was predominant (>70%, mainly during the first 24 h), less than 4% were eliminated in the feces, 2% were retained in the tissues and carcasses. Following dermal application, >50% of the dose was unabsorbed, 14-27% or <2% were respectively excreted in the urine or feces, respectively. Overall, parabens were well absorbed after oral and subcutaneous, and partially absorbed after dermal administration. All administration routes produced a single peak in the plasma, corresponding to that of para-hydroxybenzoic acid (PHBA) suggesting that PB produce no significant systemic exposure of mammalian organisms after oral, topical or subcutaneous administration.

Use of biochemical markers to monitor changes in bone turnover in cynomolgus monkeys.

The ovariectomized old cynomolgus monkey is a recognized model of human osteoporosis, and the same species can be used for the assessment of the efficacy and potential toxicity of agents intended to prevent or treat osteoporosis. Several assays have been developed that can measure the same biochemical markers of bone turnover as are used in human patients for the diagnosis and treatment follow-up of bone-related diseases, including osteoporosis. The aim of the present study was to describe the results obtained with these assays in normal control monkeys, their variations with age and sex, and their sensitivity in monitoring the bone turnover induced by ovariectomy in old skeletally mature cynomolgus monkeys. Seven old cynomolgus monkeys were bilaterally ovariectomized and 13 age-matched monkeys were sham-operated. Bone mineral density and biochemical markers were measured before and at regular intervals after surgery for up to 20 months. Total alkaline phosphatase (total ALP), bone-specific alkaline phosphatase isoenzyme (bone ALP) and osteocalcin (OC) were highly correlated to the decrease in bone mineral density (BMD) induced by ovariectomy. Deoxypyridinoline (DPD) measured by enzyme-linked immunoassay was insensitive to the bone resorption induced by ovariectomy, but cross-linked N-telopeptide (NTX-I) was higher in ovariectomized monkeys than in control monkeys. These results demonstrate that reliable biochemical parameters are available to adequately monitor and provide insight into osteoclastic bone resorption and osteoblastic bone formation, the two components of bone turnover in this animal model, and can thus be used to assess the efficacy and toxicity of potential therapeutic agents.