Measurement of genotoxicity in wastewater samples with the in vitro micronucleus test: results of a round-robin study in the context of standardisation according to ISO.

In the course of standardisation of the in vitro micronucleus test for analysis of effluents according to ISO, a national round-robin study was organised by the German Federal Institute of Hydrology (BfG), involving 10 laboratories of private companies, universities and public authorities. The micronucleus assay was performed with the permanently growing Chinese hamster lung fibroblast cell line V79. All participants tested four encoded samples from one municipal and one industrial wastewater treatment plant with and without metabolic activation by S9-mix. Two of these samples were spiked in advance with defined concentrations of the clastogenic substances cyclophosphamide and mitomycin C, respectively. Cyclophosphamide and ethyl methanesulfonate were used as positive controls. The defined assessment criterion for genotoxicity was the lowest dilution of a sample that does not show any significant induction of micronuclei. Cytotoxicity was judged by determining the cell-survival index, i.e. the percentage growth rate of the cells compared with the corresponding negative controls. As supplementary qualitative criteria, the mitotic index and the proliferation index were assessed. All participants successfully established the method within a few weeks and generated viable test results in time. The two non-genotoxic samples were detected as negative by 90% (with S9-mix) and 95% (without S9-mix) of the participants. The mitomycin C-spiked wastewater sample (expected to be positive without S9-mix supplementation) was correctly judged as positive by all laboratories. The cyclophosphamide-spiked sample (expected to be positive with S9-mix addition) was evaluated correctly as genotoxic by 80% of the laboratories. A post-test analysis found evidence that the false negative results were due to technical failure, but not of a methodological nature. In 94% of all tests the sample LID values (lowest ineffective dilution=dilution stage of the sample in the test at which a statistically significant increase in the micronucleus rate was not detectable any more) varied by no more than one dilution step around the median LID value. The survival index was proven to be a robust measure for estimation of toxicity. This round-robin study is the first inter-laboratory comparison of the in vitro micronucleus test using wastewater samples. The test system is intended to complement the already DIN- and ISO-standardised bacterial tests, i.e. the umu-test and the Ames plate-incorporation assay. The data provide evidence that the robust and practicable in vitro micronucleus test is suitable as a routine method for wastewater testing.

Tumor promoting potency of PCBs 28 and 101 in rat liver.

Only few data are available on the carcinogenic potency of individual PCB congeners. In this study, we tested the 'non-dioxinlike' congeners PCB 28 and 101 for their potency as liver tumor promoters in female rats which received diethyl-N-nitrosamine as an initiator. After 8 or 16 weeks of PCB treatment (50 and 150 micromol/kg body weight per week), each congener was recovered in the liver according to the dose levels applied, with PCB 28, at the same dose level, showing nine- to 16-fold higher hepatic levels than PCB 101 (approximately, 44 micromol/kg versus 5 micromol/kg liver at low dose, 145 micromol/kg versus 9 micromol/kg liver at high dose). PCB 28 was found to mildly induce hepatic EROD activity, while both congeners induced PROD activity. With each congener, no significant increase in the number of ATPase-deficient or GSTP-positive preneoplastic foci was obtained, while a significant increase in the relative hepatic volume of ATPase-deficient foci was found in the livers of DEN pre-treated animals having received 50 micromol/kg body weight of PCB 101 per week over 16 weeks. Our results revealed that neither the accumulative PCB 28 nor the more readily metabolisable PCB 101 was an efficacious tumor promoter in the livers of female rats.

Specific Inhibition of IkappaB kinase reduces hyperalgesia in inflammatory and neuropathic pain models in rats.

Phosphorylation of IkappaB through IkappaB kinase (IKK) is the first step in nuclear factor kappaB (NF-kappaB) activation and upregulation of NF-kappaB-responsive genes. Hence, inhibition of IKK activity may be expected to prevent injury-, infection-, or stress-induced upregulation of various proinflammatory genes and may thereby reduce hyperalgesia and inflammation. In the present study, we tested this hypothesis using a specific and potent IKK inhibitor (S1627). In an IKK assay, S1627 inhibited IKK activity with an IC50 value of 10.0 +/- 1.2 nm. In cell culture experiments, S1627 inhibited interleukin (IL)-1beta-stimulated nuclear translocation and DNA-binding of NF-kappaB. Plasma concentration time courses after intraperitoneal injection revealed a short half-life of 2.8 hr in rats. Repeated intraperitoneal injections were, therefore, chosen as the dosing regimen. S1627 reversed thermal and mechanical hyperalgesia at 3x 30 mg/kg in the zymosan-induced paw inflammation model and reduced the inflammatory paw edema at 3x 40 mg/kg. S1627 also significantly reduced tactile and cold allodynia in the chronic constriction injury model of neuropathic pain at 30 mg/kg once daily. The drug had no effect on acute inflammatory nociception in the formalin test and did not affect responses to heat and tactile stimuli in naive animals. As hypothesized, S1627 prevented the zymosan-induced nuclear translocation of NF-kappaB in the spinal cord and the upregulation of NF-kappaB-responsive genes including cyclooxygenase-2, tumor necrosis factor-alpha, and IL-1beta. Our data indicate that IKK may prove an interesting novel drug target in the treatment of pathological pain and inflammation.

Comparative proteomic analysis of the rat spinal cord in inflammatory and neuropathic pain models.

Pathological pain associated either with peripheral tissue damage and inflammation (inflammatory pain) or peripheral nerve injury (neuropathic pain) is characterized by persistent pain hypersensitivity. This hypersensitivity is believed to be mediated by sensitization of nociceptors and spinal dorsal horn neurons leading to hyperalgesia and allodynia. Changes of protein expression and/or phosphorylation are known to contribute to the development of this hyperexcitability of the nociceptive system. In the present study we analyzed protein patterns in the spinal cord following paw inflammation or sciatic nerve injury using two-dimensional (2D) gel electrophoresis combined with MALDI-TOF mass spectrometry. 2D-PAGE revealed nine and five regulated proteins following paw inflammation and sciatic nerve damage, respectively. These regulated proteins had not been identified previously with other methods. There was no overlap of regulated proteins between models except for the small heat shock protein alpha-crystallin, which was decreased in both models. In conclusion, this study illustrates that employment of the proteomic 2D-PAGE approach allows for identification of novel regulated proteins that may be involved in the central sensitization and possibly manifestation of chronic pain.

The calpain inhibitor MDL 28170 prevents inflammation-induced neurofilament light chain breakdown in the spinal cord and reduces thermal hyperalgesia.

Since long-term hyperexcitability of nociceptive neurons in the spinal cord has been suggested to be caused and maintained by changes of protein expression we assessed protein patterns in lumbar spinal cord during a zymosan induced paw inflammation employing two-dimensional (2D) gel electrophoresis. 2D PAGE revealed a time-dependent breakdown of scaffolding proteins one of which was neurofilament light chain (NFL) protein, which has been previously found to be important for axonal architecture and transport. Nociception induced breakdown of NFL in the spinal cord and dorsal root ganglias was prevented by pretreatment of the animals with a single dose of the specific inhibitor of the protease calpain (MDL-28170) which has been shown to be the primary protease involved in neurofilament degradation in neurodegenerative diseases. Treatment with the calpain inhibitor also provided anti-inflammatory and anti-hyperalgesic effects in the zymosan-induced paw inflammation model irrespective of whether the drug was administered systemically (i.p.) or delivered onto the lumbar spinal cord. This suggests that the activation of calpain is involved in the sensitization of nociceptive neurons what is partly due to neurofilament breakdown but cleavage of other calpain substrates may also be involved. Our results indicate that inhibition of pathological calpain activity may present an interesting novel drug target in the treatment of pain and inflammation.