Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.

A non-pigmented strain of Serratia marcescens (Db10) was isolated from moribund Drosophila flies. From this strain were isolated spontaneous mutants resistant to streptomycin (Db11) and nalidixic acid (Db12). Mutant Db11 was used for the isolation of two phages, phi J and phi K, which grew on Db10, Db11 and Db12, but not on three reference strains of S. marcescens. Mutant Db11 was demonstrated to fulfil koch's postulates. Strain Db10 and its antibiotic-resistant derivatives were lethal to Drosophila whether given in the food or by injection. Evidence for toxin(s) was found only in sterile supernatants from 7 d cultures. Such extracts contained proteolytic activity and inactivated the antibacterial activity in immune haemolymph from Cecropia. Phages phi J and phi K were used to isolate phage-resistant mutants of Db11. Three such mutants and their parental strain were investigated for their susceptibility to immune haemolymph from Cecropia. The parental strain was resistant to incubation with 90% haemolymph for 2 h at 37 degrees C; all phage-resistant mutants were susceptible to the immune haemolymph with "killing times" (i.e. the time required to kill 90% of the viable cells) ranging from 15 to 55 min. When the same strains were compared for their virulence to Drosophila, the phage-resistant mutants had significantly reduced virulence. It is concluded that resistance to insect immunity plays an important role in the overall pathogenicity of S. marcescens.

Fligh-throughput biomolecular proliling techniques such as transcriptomics, proteomics and metabolomics are increasingly being used in in vivo studies to recognize and characterize effects of xenobiotics on organs and systems, Of particular interest are biornalkers of treatment-related effects which are detectible in easily accessible biological fluids such as bloocl. A funclamentai challenge in such biomarker stuciies is selecting among the plethora of biomolecular changes induced by a compound and revealed by molecular profìling, to identify biomarkers which are exclusively or predominantly clLre to specific processes. In this work we present a cross-cornpaftrnent con'elation network approach, involvirig no a priori supervision or design, to integrate proteomic, metabolomic ancl transcriptomic data for selecting circulating biomarkers. The case stucly we present is the identification of bioniarkers of drug-induced hepatic toxicity effects in a rodent moclel. Biornolecular' profiling of both blood plasma and liver tissüe froni Wistar l-Iannover rats admirristerecl a toxic compound yielded many hundreds of statistically signihcant molecular changes, We exploited dmgincluced correlations between blood plasma analytes and liver tissue molecules across study animals in order to nominate selected plasnia molecules as biomarkers of drug-incluced hepatic alterations of lipid metabolisrn and urea cycle processes.

The skin tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the chlorinated insecticide, endosulfan, are two potent inhibitors of gap junctional intercellular communication. In the present study the effects of TPA and endosulfan on cell communication have been investigated in IAR 20 rat liver epithelial cells, as well as the effects of these compounds on connexin 43 (cx43), the main gap junction protein in this cell line. The results clearly demonstrate that at non-toxic doses both compounds inhibited the cell communication by at least 90% within 5 min. The communication was partially restored after 4 h of TPA exposure and almost fully restored by 24 h, whereas in endosulfan-exposed cells the communication was completely down-regulated for the whole exposure-period of 24 h. Immunoblots of IAR 20 cell extracts indicated that TPA initially caused an increased phosphorylation of cx43. A normal phosphorylation pattern was observed after 4 h when the cell communication was restored. Immunoblot analysis after endosulfan-exposure showed a slightly increased phosphorylation of cx43 after 10 min treatment, gradually followed by dephosphorylation during the rest of the 24 h treatment period. Immunostaining of IAR 20 cells showed that both compounds caused a rapid disappearance of cx43 from the cell membrane. After 4 h of exposure immunofluorescent cx43-plaques started to reappear in the cell membrane, although less pronounced in endosulfan-treated cells. However, after 24 h of endosulfan-exposure a high number of cx43-spots was demonstrated. These results indicate that different mechanisms are responsible for the inhibition of gap junctional intercellular communication induced by TPA and by endosulfan, at least during the later part of the 24 h exposure-period. TPA causes a marked hyperphosphorylation of cx43, whereas endosulfan increases phosphorylation initially only slightly but longer exposure-periods lead to hypophosphorylation. Thus, phosphorylation as well as dephosphorylation seem to be important factors involved in the regulation of the function of cx43 in this cell line.

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