Quantitative gene expression analysis in a nonhuman primate model of antibiotic-induced nephrotoxicity.

Gene expression patterns using microarrays have been described for rodent models of nephrotoxicity. To determine if significant gene expression changes previously identified have application across multiple species, we studied quantitative gene expression changes in the kidneys of female cynomolgus monkeys after exposure to two nephrotoxicants. Animals were dosed with the aminoglycoside gentamicin (10 mg/kg), the experimental oligosaccharide antibiotic everninomicin (30 or 60 mg/kg), or a combination of gentamicin (10 mg/kg) and everninomicin (30 mg/kg) for 7 days. Monkeys receiving these drugs in combination developed renal lesions as early as Day 1. By Day 7, monkeys dosed with 60 mg/kg everninomicin alone also developed renal lesions, while the group exposed to both compounds had more extensive renal damage. The modulation of several genes previously reported to be associated with nephrotoxicity in rodent models was confirmed using quantitative real-time PCR. Among these, waf-1, matrix metalloproteinase-9, and vimentin exhibited changes consistent with the definition of a genomic indicator of toxicity. In addition, we identified three early gene biomarkers that may be predictive of drug-induced nephrotoxicity: clusterin, osteopontin, and hepatitis A virus cellular receptor-1. Logistic regression demonstrated a high degree of correlation between changes in gene expression and the probability of the development of histopathologic lesions. These results are the first confirming rodent gene expression changes associated with nephrotoxicity in a nonhuman primate model and provide preliminary evidence for identifying early gene expression changes predicting the onset of drug-induced renal tubular damage in cynomolgus monkeys.

Assessment of the sensitivity of the computational programs DEREK, TOPKAT, and MCASE in the prediction of the genotoxicity of pharmaceutical molecules.

Computational models are currently being used by regulatory agencies and within the pharmaceutical industry to predict the mutagenic potential of new chemical entities. These models rely heavily, although not exclusively, on bacterial mutagenicity data of nonpharmaceutical-type molecules as the primary knowledge base. To what extent, if any, this has limited the ability of these programs to predict genotoxicity of pharmaceuticals is not clear. In order to address this question, a panel of 394 marketed pharmaceuticals with Ames Salmonella reversion assay and other genetic toxicology findings was extracted from the 2000-2002 Physicians' Desk Reference and evaluated using MCASE, TOPKAT, and DEREK, the three most commonly used computational databases. These evaluations indicate a generally poor sensitivity of all systems for predicting Ames positivity (43.4-51.9% sensitivity) and even poorer sensitivity in prediction of other genotoxicities (e.g., in vitro cytogenetics positive; 21.3-31.9%). As might be expected, all three programs were more highly predictive for molecules containing carcinogenicity structural alerts (i.e., the so-called Ashby alerts; 61% +/- 14% sensitivity) than for those without such alerts (12% +/- 6% sensitivity). Taking all genotoxicity assay findings into consideration, there were 84 instances in which positive genotoxicity results could not be explained in terms of structural alerts, suggesting the possibility of alternative mechanisms of genotoxicity not relating to covalent drug-DNA interaction. These observations suggest that the current computational systems when applied in a traditional global sense do not provide sufficient predictivity of bacterial mutagenicity (and are even less accurate at predicting genotoxicity in tests other than the Salmonella reversion assay) to be of significant value in routine drug safety applications. This relative inability of all three programs to predict the genotoxicity of drugs not carrying obvious DNA-reactive moieties is discussed with respect to the nature of the drugs whose positive responses were not predicted and to expectations of improving the predictivity of these programs. Limitations are primarily a consequence of incomplete understanding of the fundamental genotoxic mechanisms of nonstructurally alerting drugs rather than inherent deficiencies in the computational programs. Irrespective of their predictive power, however, these programs are valuable repositories of structure-activity relationship mutagenicity data that can be useful in directing chemical synthesis in early drug discovery.

Quantitative PCR deconstruction of discrepancies between results reported by different hybridization platforms.

Differences in hybridization platforms used in gene array analysis experiments can lead to significant differences in hybridization results. In this study we used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to investigate discrepant results between the National Institute of Environmental Health Sciences cDNA and Affymetrix oligo platforms used to evaluate hepatic gene expression changes in rats exposed to methapyrilene. Caldesmon cDNA platform hybridization results showed decreases in gene expression levels for the high-dose methapyrilene 7-day pooled samples compared with their controls. By contrast, the Affymetrix oligonucleotide platform showed increases in expression levels for these samples. Quantitative gene expression measurements provide an explanation for the discrepancies observed for these samples. In the case of caldesmon, there is a 74-base sequence in the cDNA clone that is absent in the Affymetrix sequence. The amplicon based on the cDNA clone shows > 100-fold suppression relative to the day 7 high-dose methapyrilene-pooled control. These data demonstrate the importance of using a "gold standard," such as qRT-PCR to confirm key hybridization results as well as to understand the sources of discrepancies resulting from different hybridization platforms.

Assessment of temporal biochemical and gene transcription changes in rat liver cytochrome P450: utility of real-time quantitative RT-PCR.

PURPOSE: A conventional approach to assess cytochrome P450 (CYP) induction in preclinical animal models involves daily dosing for a least a week followed by Western blot and/or enzyme activity analysis. To evaluate the potential benefit of a third more specific and sensitive assay, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), with the objective of reducing the duration of the conventional 1-week study, we simultaneously assessed gene expression by qRT-PCR along with Western blots and enzyme activity assays as a time course in an in vivo model. METHODS: Rats were dosed daily for 8 days with model inducers of CYP1A, CYP2B, CYP3A, or CYP4A. Liver P450 levels were measured after 0.5, 1, 2, 4, and 8 days of dosing by qRT-PCR, Western blot, and enzyme activity. RESULTS: CYP1A, CYP3A, and CYP4A genes were maximally induced very rapidly (0.5-1 day), whereas the CYP2B gene was maximally induced after a lag time of 4 days. In all cases, fold changes in induction detected by qRT-PCR were greater than fold changes in protein levels and enzyme activities. CONCLUSIONS: Maximal persistent and larger fold changes observed by qRT-PCR either preceded or occurred simultaneously with maximal sustained fold changes in protein levels as measured by Western blots and enzyme activity assays. Our data show that qRT-PCR provides increased sensitivity and specificity over conventional assays and may be key information for reliable assessment of drug-related changes in CYP induction during the transition from discovery to toxicology studies.

Carcinogenicity testing of IL-10: principles and practicalities.

IL-10 is a cytokine with actions at many levels of the immune system. In the course of development of recombinant human IL-10 (rhuIL-10) as a potential treatment for a number of chronic diseases of man, the question 'What about its carcinogenicity testing?' was repeatedly asked, based on scientific evaluation by toxicologists, beliefs about regulatory requirements, and questions considered likely to be raised by physicians, patients, and lawyers. The feasibility of various approaches to the carcinogenicity testing of rhuIL-10 is critically discussed here as a contribution to rational consideration of the general need for and value of such testing, and its particular feasibility for a recombinant human protein with profound effects on the immune system. The physiological functions of IL-10 in man and rodents are reviewed in detail, as there are notable differences between species in its normal activities, followed by detailed evaluation of the potential procedures and practical problems of its carcinogenicity testing as a heterologous, immunogenic protein in rodents. The value of information that might be obtained from transgenic mice is also evaluated, and so are the results of studies exploring its actions on human tumour cell biopsies and rodent and human cell lines. It is concluded that despite the probable popular and regulatory expectations that carcinogenicity test results would be provided, all the physiological and pathological information reveals no indication that rhuIL-10 would pose a carcinogenic risk to humans on prolonged administration, and that it would not be feasible to undertake such experimentation. It is argued that in this, as in other instances, professional and popular expectations have run beyond practical feasibility or theoretical justification. Cautious and critical evaluation should be made every time shorter or longer term toxicity studies of any candidate drug are planned or even considered, especially if it is a recombinant protein, to decide on objective grounds whether the studies are really necessary and whether they can be done in a way that will give meaningful results that will help in risk assessment.

Preclinical safety evaluation of recombinant human interleukin-10.

Escherichia coli-derived recombinant human interleukin-10 (rhuIL-10) has been evaluated in an extensive series of in vivo and in vitro nonclinical safety studies, including genetic toxicology, single- and repeat-dose systemic toxicity and toxicokinetics, reproductive toxicity, and specialized irritation studies. The primary test species in the toxicology studies were the mouse and monkey based on rhuIL-10 activity in receptor binding and ex vivo cytokine assays. Supported by a detailed preclinical program of therapeutic and prophylactic animal models in autoimmune diseases, the initial clinical development program has focused on investigating the therapeutic potential of rhuIL-10 (Tenovil) in Crohn's disease and rheumatoid arthritis. The results of the subcutaneous toxicity studies, up to 3 months dosing duration in mice and 6 months dosing duration in monkeys, support the development of rhuIL-10 for present and future clinical indications by the subcutaneous route of administration.

Safety evaluation of recombinant human interleukin-4. I. Preclinical studies.

Recombinant human IL-4 (rhuIL-4) has been evaluated in a series of preclinical studies. These studies have demonstrated that rhuIL-4 is a very potent cytokine with a wide range of pharmacologic and toxicologic effects. Target systems/organs included the cardiovascular system, liver, spleen, and bone marrow. The incidence and severity of effects correlated strongly with both the dose level and the duration of rhuIL-4 administration. The major dose-limiting toxicities identified included death, cardiac inflammation and necrosis, hepatitis, and hepatic necrosis and occurred at sc doses > or = 25 micrograms/kg/day, while a sc dose of 5 micrograms/kg/day was the highest tested that did not result in major dose-limiting toxicity. Clinical trials in humans have demonstrated that sc administration of Escherichia coli-derived rhuIL-4 is safe and well tolerated at doses up to and including 5 micrograms/kg/day and up to 10 micrograms/kg when administered 3 times/week.

Safety evaluation of recombinant human interleukin-4. II. Clinical studies.

The safety and tolerability of Escherichia coli-derived recombinant human interleukin-4 (rhuIL-4) have been evaluated in phase I and phase II studies in human patients with a variety of malignancies. Clinical trials have demonstrated that subcutaneous administration of rhuIL-4 is safe and well tolerated at doses as high as 5 micrograms/kg/day and as high as 10 micrograms/kg when administered 3 times/week. Although preclinical safety studies in cynomolgus monkeys demonstrated a number of adverse effects following repeated daily dosing with rhuIL-4, similar effects have generally not been observed in human patients.

DNA double-strand damage and repair following gamma-irradiation in isolated spermatogenic cells.

Various cell types in spermatogenesis exhibit differential sensitivity to radiation-induced DNA damage. The investigation of DNA radiosensitivity in vitro is complicated by the heterogeneous population of male germ cells (MGC) present in isolated single-cell suspensions. In the present investigation, the neutral elution technique was used to assess gamma-irradiation-induced DNA double-strand damage (DSD) in spermatogonia and preleptotene spermatocytes (SG/PL), pachytene spermatocytes and spermatid spermatocytes, as well as in MGC. In addition, the capability of these cell types to repair DNA double-strand damage was investigated. Based on the well established timing of the rat spermatogenic cycle, the DNA of specific cell populations was labeled using tritiated thymidine. DNA from labeled cells was determined isotopically, whereas total DNA was quantitated using a fluorometric method. DSD was induced in a dose-dependent manner in the heterogeneous population as well as in the labeled cell populations. SG/PL were more sensitive to gamma-irradiation-induced DSD than either the heterogeneous MGC population, pachytene or spermatid spermatocytes. Each cell type exhibited a similar capability to repair DSD following exposure to 3000 rad; repair was rapid (maximal within 45 min) and incomplete (less than 40%). Only pachytene spermatocytes exhibited significant repair following exposure to 6000 rad. Since a difference in sensitivity to radiation-induced DSD was demonstrated, the capability of each cell type to repair a similar initial frequency of strand damage was investigated. SG/PL, pachytene and spermatid spermatocytes differed in their capability to repair similar levels of strand damage. However, the difference in dose required to achieve equal damage may have contributed to other cellular effects, thus altering repair. In summary, a model is described that permits the evaluation of genotoxic responses in specific populations of spermatogenic cells within a heterogeneous cell suspension. The ability of specific cell types to repair gamma-irradiation-induced DNA double-strand damage is demonstrated.

Autoradiographic evidence for insulin and insulin-like growth factor binding to early mouse embryos.

Insulin binding to mouse oocytes and preimplantation embryos was assessed by light-microscopic autoradiography. Significant insulin binding was present on the cells of morulae and increased twofold at the blastocyst stage of development. Insulin binding was markedly decreased by native insulin and to a lesser extent by insulin-like growth factors (IGFs). No specific insulin binding was detected on oocytes or embryos throughout the eight-cell stage. Specific binding of IGF-I and IGF-II was also observed on the cells of blastocyst outgrowths. The findings demonstrate that specific binding of insulin and IGF is temporally expressed on the cells of pre- and peri-implantation mouse embryos. These results confirm and extend our previous immunofluorescence study.

Bleomycin-induced DNA-strand damage in isolated male germ cells.

DNA strand damage in isolated male germ cells (MGC) was evaluated after in vitro exposure to bleomycin (BLM), a known genotoxin. The alkaline elution technique was used to determine DNA-strand breaks. Concentration-dependent strand damage was established following exposure to bleomycin for 1 h at 37 degrees C. Exposure at 0 degrees C resulted in an increase in the frequency of strand breaks as compared to those observed at 37 degrees C. Pretreatment of cells with deferoxamine (DM), an iron-selective chelating agent, abolished the DNA damage induced by bleomycin. Isolated male germ cells responded in a predictable and reproducible manner thus supporting their use in mechanistic studies of genotoxicity.

Stage-specific insulin binding in mouse preimplantation embryos.

Stage-specific insulin binding in the developing mouse embryo was demonstrated by an indirect immunofluorescence technique using an antibody against insulin. Concentration-dependent fluorescence labeling was observed in the morula and blastocyst stages of development, whereas no reactivity was seen in unfertilized oocytes or in 2-, 4-, and 8-cell embryos. The possible significance of these observations is discussed. This represents the first report of stage-specific insulin binding during mammalian preimplantation development.

Impaired renal function in diabetic chimpanzees (Pan troglodytes).

Studies were conducted to assess the renal functional state in two recently discovered diabetic chimpanzees. Both were nonobese, adult female animals with the non-insulin-dependent form of impaired glucose tolerance, analogous to the Type II or nonobese, maturity-onset diabetes of humans. Both animals displayed moderate-to-heavy proteinuria and glycosuria in response to intravenous administration of glucose or tolbutamide. Chimpanzee number 333, but not number 1037, had fasting proteinuria and chronic hypertension. Renal function studies, using the inulin clearance method, demonstrated significantly decreased glomerular filtration rates and elevated rates of sodium excretion for both animals. The rate of chloride excretion was also elevated in animal number 1037, but potassium excretion was apparently unaffected in both animals. Abnormal serum biochemical parameters demonstrated for chimpanzee number 333 included elevations in calcium, magnesium, creatinine, urea nitrogen, and uric acid; animal number 1037 had only an elevated serum creatinine. Results are consistent with the occurrence of renal disease similar to the nephropathy that develops in human diabetics. The difference in severity of renal impairment in the two chimpanzees is possibly related to differences in duration and severity of impaired glucose tolerance. A progression of both diabetic and renal disorders is most probable.

Diabetes mellitus in the chimpanzee (Pan troglodytes).

Glucose intolerance was found in four adult chimpanzees. The response of glucose, insulin, C-peptide, and glucagon to intravenous glucose and tolbutamide stimulations revealed impaired glucose clearance, deficient pancreatic secretion of insulin and C-peptide, and elevated glucagon levels. Pancreatic islets in a diabetic chimpanzee were hypercellular, possible due to alpha-cells. Minimal or no insulin was observed in beta-cells. Results are consistent with the occurrence of noninsulin-dependent diabetes mellitus, which may be more prevalent in chimpanzees than heretofore suspected.

Chemically induced (streptozotocin-alloxan) diabetes mellitus in the dog. Biochemical and ultrastructural studies.

Adult beagle dogs were infused intravenously with alloxan (50 mg/kg) and streptozotocin (30 mg/kg) in order to investigate sequential changes in plasma glucose, insulin, glucagon, and cortisol. These biochemical findings were correlated with ultrastructural alterations in pancreatic islets. Following infusion, the dogs became hyperglycemic by 2 hours, severely hypoglycemic by 6 to 14 hours, and permanently hyperglycemic by 24 hours. Plasma immunoreactive insulin increased sharply from 6 to 10 hours, then declined to nearly undetectable levels by 24 hours. Ultrastructurally, beta cells at 2 hours had clumped nuclear chromatin, vacuolated mitochondria, and dilated individual profiles of endoplasmic reticulum. Secretory granules appeared swollen but retained their internal cores. Degeneration of beta cells was severe after 10 hours, and plasma membranes of beta cells were disrupted by 24 hours. The cytoplasmic area of adjacent beta cells coalesced and had accumulations of membranous debris, lipofuscin, and autophagic vacuoles. Alpha and delta cells appeared to be unaffected. Plasma glucagon levels decreased markedly at 10 hours and were related reciprocally to changes in plasma insulin. Pancreatic islets in dogs with chronic (16 months) diabetes were small and composed primarily of granulated alpha and delta cells. Poorly granulated beta cells with degenerative changes were present in an occasional islet. The results of this investigation demonstrated that the combined administration of a single dose of alloxan and streptozotocin selectively destroyed the beta cells, while alpha and delta cells of the islets of Langerhans remained unaltered. Pathologic evidence of toxicity was not present in other organs. Chemically induced diabetes mellitus in dogs is a reproducible animal model that should prove useful in studies requiring repeated experimental manipulations or sampling of biologic fluids in order to evaluate the long-term effects of different routes of delivery or preparations of insulin to control the persistent hyperglycemia.