Genotoxicity studies of a desealant solvent mixture, SR-51.

The Royal Australian Air Force (RAAF) has reported that personnel involved in F-111 fuel tank maintenance were concerned that exposure to a range of chemicals during the period 1977 to mid-1990s was the cause of health problems, including cancer. Particular concern was directed at SR-51, a desealant chemical mixture containing the following four solvents: aromatic 150 solvent (Aro150), dimethylacetamide, thiophenol (TP), and triethylphosphate. The present study examined the mutagenic potential of SR-51 using a range of well-known mutagen and genotoxin assays. The tests used were i) a modified version of the Ames test, ii) the mouse lymphoma assay, iii) the comet assay (a single-cell gel electrophoresis assay), and iv) a mouse micronucleus test. The modified Ames test used mixed bacterial strains in liquid suspension media. The Ames test results showed that SR-51 (tested up to the cytotoxic concentration of 36 microg/ml, 30 min incubation) in the presence and absence of S9 metabolic activation was not mutagenic. The mouse lymphoma assay used cultured mouse lymphoma cells in a microwell suspension method. The mouse lymphoma assay was also negative with SR-51 (tested up to the cytotoxic concentration of 22.5 microg/ml, 3 h incubation) in the presence and absence of S9 metabolic activation. The Comet assay, using cultured mouse lymphoma cells, showed no evidence of DNA damage in cells exposed up to the cytotoxic concentration of SR-51 at 11.25 microg/ml. The in-vivo mouse micronucleus test was undertaken in wild-type C57Bl6J male mice dosed orally with SR-51for 14 days with a single daily dose up to 360 mg/kg/day (the maximum-tolerated dose). No increases were observed in micronuclei (MN) frequency in bone marrow collected (24 h after final dose) from SR-51-treated mice compared to the number of MN observed in bone marrow collected from untreated mice. Tissues collected from treated mice at necropsy demonstrated a significant increase in spleen weights in the high dose mice. Gas chromatography analysis of SR-51 identified more than 40 individual components and an oxidation product, diphenyldisulfide derived from TP under conditions of mild heating. In conclusion, there was no evidence that SR-51 is mutagenic.

Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture.

BACKGROUND: The traditional anesthetic used for collection of the serum culture medium for whole rat embryo culture studies has been ether. However ethical concerns have been raised due to the irritant nature of the vapour and safety concerns due to the risk of fire. METHODS: Growth and development of gestation day 9.5 rat embryos cultured for 48 h in serum collected from rats anesthetised with either ether, isoflurane or halothane were compared. RESULTS: There were no differences in any of the parameters used to assess embryonic development when embryos were grown in serum collected using either ether or isoflurane anesthetics. However, when embryos grown in serum collected using ether or halothane were compared, embryonic development was similar in all respects, except for a reduced number of embryos turned to become fully dorsally convex in the halothane group (p <0.05). CONCLUSIONS: The data indicate that isoflurane is an appropriate alternative to ether for collection of the serum culture medium for whole rat embryo culture, while halothane may cause some delay of embryonic development.

Testicular changes induced by chronic exposure to the herbicide formulation, Tordon 75D (2,4-dichlorophenoxyacetic acid and picloram) in rats.

The second most used herbicide in the Vietnam war was Agent White, which contained the active components 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram). The herbicide formulation Tordon 75D is similar in terms of its active components to Agent White and is currently used by the agricultural industry in Australia. As part of an investigation into the possible adverse effects of this herbicide on male reproductive performance, groups of five male rats were gavaged 5 days a week for 9 weeks with either 0.125 ml/kg (low dose), 0.25 ml/kg (middle dose), or 0.5 ml/kg (high dose) Tordon 75D or water (controls). The high dose corresponded to 150 mg/kg body weight 2,4-D and 37.5 mg/kg picloram acid equivalents. At the end of the treatment period, the testes were collected, weighed, and examined histologically and blood samples were taken to determine serum testosterone. Groups of high dose animals were also examined after 1, 2, and 4 weeks treatment. The 9 weeks treatment with Tordon 75D caused severe reduction in testicular weight in some high dose animals. Histologically, the small testes showed shrunken tubules with germ cell depletion. This damage was still evident in some rats following a 21 weeks recovery period suggesting that the testicular damage was permanent. Testicular damage was not due to endocrine disruption as there were no significant differences in the serum concentration of testosterone in control animals compared to Tordon 75D-treated animals. Blood levels associated with the high dose were determined in a separate study and were much higher than those likely to be obtained by occupational exposure to this herbicide.

A study of the potential for a herbicide formulation containing 2,4-d and picloram to cause male-mediated developmental toxicity in rats.

Male Vietnam veterans have repeatedly expressed concern that exposure to herbicides in Vietnam may have caused birth defects in their offspring. The second most used herbicide was a mixture of 2,4-D and picloram called Agent White. This study is an investigation into the possible male-mediated reproductive toxicology of this herbicide. Male rats were gavaged for 5 days per week for 9 weeks with a mixture of 2,4-D and picloram called Tordon 75D(R) (the Australian derivative of Agent White). Three doses were tested; the high dose was considered the maximum tolerated dose. Each male was mated with two untreated females during weeks 2 and 3, 4 and 5, and 8 and 9 of treatment, and with four untreated females after an 11-week recovery period. Negative controls were males dosed with distilled water, and positive controls were males dosed with cyclophosphamide at 5.1 mg/kg/day. All mated females were killed on day 20 of gestation, and the fetuses were weighed and examined for either structural malformations or skeletal development. Litter size, fetal weight, and malformation rate were all unaffected by treatment. The cyclophosphamide positive controls showed the expected large increase in postimplantation loss. In general, within the limitations of the power of the study, the results did not show any evidence that exposure to a herbicide formulation containing 2,4-D and picloram is likely to cause male-mediated birth defects or other adverse reproductive outcomes.

The use of short-form quality of life questionnaires to measure the impact of imipramine on women with urge incontinence.

Short-form questionnaires were used to measure the change in quality of life (QOL) of women with urge-predominant urinary incontinence treated with imipramine hydrochloride. Short forms of the Incontinence Impact Questionnaire (IIQ-7) and the Urogenital Distress Index (UDI-6) were integrated into a patient questionnaire, which was given to 25 patients with urge-predominant urinary incontinence before and after treatment with imipramine. Demographic data and self-reports of the number of incontinent episodes were also recorded. Total and subscale QOL scores and number of incontinent episodes were recorded and compared with Wilcoxson's signed ranks test, as well as correlated to the change in number of incontinent episodes with Pearson's correlation coefficient. Treatment with imipramine resulted in a clinical improvement or cure in 16/22 patients (72.7%), with an average reduction in incontinent episodes of 78.7% (P<0.001). The average per cent improvement in QOL scores for total IIQ-7 was 42.1% (P<0.01) and total UDI-6 score was 44.1% (P<0.001). All subscale QOL differences were also significant (P<0.01). The incidence of side effects to imipramine was 41%, which resulted in dose changes. Fourteen per cent eventually discontinued therapy. Neither total nor subscale QOL improvement scores were correlated with improvement in number of incontinent episodes. The short form IIQ-7 and UDI-6 are effective tools to determine change in QOL, as evidenced by the effectiveness of imipramine for the treatment of urge-predominant urinary incontinence. Significant reductions in incontinent episodes and improvements in IIQ-7 and UDI-6 QOL scores were both seen, but were not correlated. Short-form QOL measures can easily be integrated into a patient questionnaire to objectively measure a very subjective topic.

Apoptotic cleavage of cytoplasmic dynein intermediate chain and p150(Glued) stops dynein-dependent membrane motility.

Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.

Phosphorylation by cdc2-CyclinB1 kinase releases cytoplasmic dynein from membranes.

Movement of various cargoes toward microtubule minus ends is driven by the microtubule motor cytoplasmic dynein (CD). Many cargoes are motile only during certain cell cycle phases, suggesting that CD function may be under cell cycle control. Phosphorylation of the CD light intermediate chain (DLIC) has been suggested to play a crucial role in modulating CD function during the Xenopus embryonic cell cycle, where CD-driven organelle movement is active in interphase but greatly reduced in metaphase. This down-regulation correlates with hyperphosphorylation of DLIC and release of CD from the membrane. Here we investigate the role of the key mitotic kinase, cdc2-cyclinB1, in this process. We show that DLIC within the native Xenopus CD complex is an excellent substrate for purified Xenopus cdc2-glutathione S-transferase (GST) cyclinB1 (cdc2-GSTcyclinB1) kinase. Mass spectrometry of native DLIC revealed that a conserved cdc2 site (Ser-197) previously implicated in the metaphase modulation of CD remains phosphorylated in interphase and so is unlikely to be the key regulatory site. We also demonstrate that incubating interphase membranes with cdc2-GSTcyclinB1 kinase results in substantial release of CD from the membrane. These data suggest that phosphorylation of DLIC by cdc2 kinase leads directly to the loss of membrane-associated CD and an inhibition of organelle movement.

Therapeutic benefit of a dissociated glucocorticoid and the relevance of in vitro separation of transrepression from transactivation activity.

Glucocorticoids (GCs) are the mainstay of asthma therapy; however, major side effects limit their therapeutic use. GCs influence the expression of genes either by transactivation or transrepression. The antiinflammatory effects of steroids are thought to be due to transrepression and the side effects, transactivation. Recently, a compound, RU 24858, has been identified that demonstrated dissociation between transactivation and transrepression in vitro. RU 24858 exerts strong AP-1 inhibition (transrepression), but little or no transactivation. We investigated whether this improved in vitro profile results in the maintenance of antiinflammatory activity (evaluated in the Sephadex model of lung edema) with reduced systemic toxicity (evaluated by loss in body weight, thymus involution, and bone turnover) compared with standard GCs. RU 24858 exhibits comparable antiinflammatory activity to the standard steroid, budesonide. However, the systemic changes observed indicate that transactivation events do occur with this GC with similar potency to the standard steroids. In addition, the GCs profiled showed no differentiation on quantitative osteopenia of the femur. These results suggest that in vitro separation of transrepression from transactivation activity does not translate to an increased therapeutic ratio for GCs in vivo or that adverse effects are a consequence of transrepression.