An alternative linear trend analysis for assessing the results of the mouse lymphoma assay.

As recommended by the mouse lymphoma assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing (Aberdeen, 2003), a trend test is critical if an induced mutant frequency (MF) of at least 126 × 10(-6) (global evaluation factor, GEF) is achieved at one or more test concentrations. Only those responses that both achieve the GEF and a significant trend are biologically relevant. While no specific trend test was recommended by the Workshop, a trend test was recommended by the UK Environmental Mutagen Society (1989). The test uses MF (untransformed) averaged over replicate cultures following a consistency test (against a historical heterogeneity factor) in a weighted linear regression with chi-square (χ(2)) test for slope and returns significant results in virtually all cases that are positive for the GEF, including those with no apparent dose-response. We have explored an alternative method where the natural logarithm of MF and its variance are estimated for each replicate culture separately and used in a weighted ordinary linear regression with t-test for slope. Using test cases positive for the GEF, the P-value from this model is shown to be sensitive to changes in the number of replicates, the shape and magnitude of mutant induction, in contrast to the χ(2) model. Cases with no apparent dose-response and thereby questionable biological significance are tested negative by our method but positive by the χ(2) model. Our method is thus straight-forward and provides a meaningful complement to the GEF in assessing the biological significance of the MLA results.

Tesaglitazar, a dual PPAR-α/γ agonist, hamster carcinogenicity, investigative animal and clinical studies.

Tesaglitazar was developed as a dual peroxisome proliferator-activated receptor (PPARα/γ). To support the clinical program, a hamster carcinogenicity study was performed. The only neoplastic findings possibly related to treatment with tesaglitazar were low incidences of hemangioma and hemangiosarcoma in the liver of male animals. A high-power, two-year investigative study with interim necropsies was performed to further elucidate these findings. Treatment with tesaglitazar resulted in changes typical for exaggerated PPARα pharmacology in rodents, such as hepatocellular hypertrophy and hepatocellular carcinoma, but not an increased frequency of hemangiosarcomas. At the highest dose level, there was an increased incidence of sinusoidal dilatation and hemangiomas. No increased endothelial cell (EC) proliferation was detected in vivo, which was confirmed by in vitro administration to ECs. Immunohistochemistry and gene expression analyses indicated increased cellular stress and vascular endothelial growth factor (VEGF) expression in the liver, which may have contributed to the sinusoidal dilatation. A two-fold increase in the level of circulating VEGF was detected in the hamster at all dose levels, whereas no effect on VEGF was observed in patients treated with tesaglitazar. In conclusion, investigations have demonstrated that tesaglitazar does not produce hemangiosarcomas in hamster despite a slight effect on vascular morphology in the liver.

Structural and functional differences between neuropathy with and without pain?

We aimed to find functional and structural differences in neuropathy between patients with and without chronic pain following nerve injury. We included 30 patients requiring hand surgery after a trauma, with 21 reporting chronic pain for more than one year after the injury, while 9 did not suffer from injury-related chronic pain. We assessed mechanical sensitivity, thermal thresholds, electrically induced pain and axon reflex erythema and cutaneous nerve fiber density in skin biopsies of the injured site and its contralateral control. Epidermal fiber density of the injured site was reduced similarly in both patient groups. Thresholds for cold and heat pain and axon reflex areas were reduced in the injured site, but did not differ between the patient groups. Only warmth thresholds were better preserved in the pain patients (35.2 vs. 38.4°C). Neuronal CGRP staining did not reveal any difference between pain and non-pain patients. Epidermal innervation density correlated best to warmth detection thresholds and deeper dermal innervation density to the area of the axon reflex erythema. No specific pattern of subjective, functional or structural parameters was detected that would separate the neuropathy patients into pain and non-pain patients. Specific staining of additional targets may help to improve our mechanistic understanding of pain development.

Tesaglitazar, a PPARalpha/gamma agonist, induces interstitial mesenchymal cell DNA synthesis and fibrosarcomas in subcutaneous tissues in rats.

The development of the dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year carcinogenicity study in rats given 0.3, 1, 3, and 10 micromol/kg tesaglitazar is presented with focus on the findings of subcutaneous fibrosarcomas. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis (immunohistochemical detection of BrdU-labeled cells) and expression of PPARgamma (immunohistochemistry and reverse transcription-polymerase chain reaction) in subcutaneous adipose tissues was assessed in rats administered 1 or 10 micromol/kg for 2 weeks or 3 months. Poorly differentiated subcutaneous mesenchymal sarcomas with a predominant spindle cell appearance occurred at the highest dose level of 10 micromol/kg in both sexes, and these tumors were diagnosed as fibrosarcomas. The 10-micromol/kg dose was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased at 1 and 10 micromol/kg after 2 weeks. The increase in DNA synthesis was still significant at the end of the 12-week treatment at 10 mumol/kg, the dose producing fibrosarcoma. However, at 1 micromol/kg, a dose below the no-observed-effect level for fibrosarcoma, the level of DNA synthesis was similar to control levels at 12 weeks. Immunohistochemical analyses showed no detectable PPARgamma protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARgamma in these cells. The results suggest that the induction of rat fibrosarcoma by tesaglitazar, at exposures 100-fold above the human therapeutic exposure, may involve proliferation of undifferentiated mesenchymal cells in subcutaneous tissues.