Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.     

Quantitative analysis of N-terminal valine peptide adducts specific for 1,2-epoxy-3-butene

Butadiene (BD) metabolism shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is metabolized mainly by cytochrome P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. Analysis of N-terminal globin adducts is a common approach for monitoring the internal formation of BD derived epoxides. Our long term strategy is to develop an LC-MS/MS method for simultaneous detection of all three BD hemoglobin adducts. This approach is modeled after the recently reported immunoaffinity LC-MS/MS method for the cyclic N,N-(2,3-dihydroxy-1,4-butadyil)-valine (pyr-Val, derived from DEB). We report herein the analysis of the EB-derived 2-hydroxyl-3-butenyl-valine peptide (HB-Val). The procedure utilizes trypsin hydrolysis of globin and immunoaffinity (IA) purification of alkylated heptapeptides. Quantitation is based on LC-MS/MS monitoring of the transition from the singly charged molecular ion of HB-Val (1-7) to the a(1) fragment. Human HB-Val (1-11) was synthesized and used for antibody production. As internal standard, the labeled rat-[(13)C(5)(15)N]-Val (1-11) was prepared through direct alkylation of the corresponding peptide with EB. Standards were characterized and quantified by LC-MS/MS and LC-UV. The method was validated with different amounts of human HB-Val standard. The recovery was >75% and coefficient of variation <25%. The LOQ was set to 100 fmol/injection. For a proof of principal experiment, globin samples from male and female rats exposed to 1000 ppm BD for 90 days were analyzed. The amounts of HB-Val present were 268.2+/-56 and 350+/-70 pmol/g (mean+/-S.D.) for males and females, respectively. No HB-Val was detected in controls. These data are much lower compared to previously reported values measured by GC-MS/MS. The difference may be due higher specificity of the LC-MS/MS method to the N-terminal peptide from the alpha-chain versus derivatization of both alpha- and beta-chain by Edman degradation, and possible instability of HB-Val adducts during long term storage (about 10 years) between the analyses. These differences will be resolved by examining recently collected samples, using the same internal standard for parallel analysis by GC-MS/MS and LC-MS/MS. Based on our experience with pyr-Val adduct assay we anticipate that this assay will be suitable for evaluation of HB-Val in multiple species.     

Positive short-term effects of sheep grazing on the alpine avifauna

Grazing by large herbivores may negatively affect bird populations. This is of great conservation concern in areas with intensive sheep grazing. Sheep management varies substantially between regions, but no study has been performed in less intensively grazed systems. In a fully replicated, landscape scale experiment with three levels of sheep grazing, we tested whether the abundance and diversity of an assemblage of mountain birds were negatively affected by grazing or if grazing facilitated the bird assemblage. Density of birds was higher at high sheep density compared with low sheep density or no sheep by the fourth grazing season, while there was no clear effect on bird diversity. Thus, agricultural traditions and land use politics determining sheep density may change the density of avifauna in either positive or negative directions.     

Targeted proteomics on its way to discovery

For a long time, targeted and discovery proteomics covered different corners of the detection spectrum, with targeted proteomics focused on small target sets. This changed with the recent advances in highly multiplexed analysis. While discovery proteomics still pushes higher numbers of identified and quantified proteins, the advances in targeted proteomics rose to cover large parts of less complex proteomes or proteomes with low protein detection counts due to dynamic range restrictions, like the blood proteome. These new developments will impact, especially on the field of biomarker discovery and the possibility of using targeted proteomics for diagnostic purposes.     

Lack of evidence for an immunosuppressive role for MUC1

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1- and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells. Received: 20 August 1998 / Accepted: 3 December 1998     

Impact of drug resistance on fitness of Mycobacterium tuberculosis strains of the W-Beijing genotype

Mycobacterium tuberculosis strains of the W-Beijing genotype became a common cause of tuberculosis during the past years and they are often associated with drug resistance. The biological factors facilitating the selection and wide dissemination of these strains are not known. To determine how acquisition of drug resistance affected growth of strains of the W-Beijing genotype, the growth of 55 M. tuberculosis isolates were studied using the BBL MGIT Mycobacteria Growth Indicator Tube and the BACTEC MGIT 960 System. Susceptible strains of non-Beijing genotypes were found to be the most fit strains. Drug-resistant strains of non-Beijing genotypes were more likely to grow slower than susceptible strains (P=0.001). Drug-resistant strains of the W-Beijing genotype had two tendencies of growth: some of them showed reduced growth compared to susceptible strains, while others did not show loss of fitness measured as growth.