Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

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.     

Thyroxine,methimazole, and thyroid microsomal autoantibody titres in hypothyroid Hashimoto's thyroiditis.

Ten hypothyroid patients with Hashimoto''s thyroiditis were treated with methimazole 30 mg in addition to thyroxine 0.15 mg daily. Another 10 hypothyroid patients with Hashimoto''s thyroiditis were given thyroxine 0.15 mg alone. After 22 weeks of treatment significant decreases in thyroid microsomal autoantibody titres were observed in both groups (p less than 0.01). There was no difference in the mean change in titre between the two groups. When the patients treated with methimazole were subsequently given thyroxine 0.15 mg alone for a further 22 weeks no additional change in titre was observed. The data suggest that thyroxine, by normalising serum thyroid stimulating hormone concentrations, may reduce the autoantigenic properties of the thyrocytes with a subsequent decrease in autoantibody titres.     

A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants

We propose a nomenclature for the genes encoding the chlorophylla/b-binding proteins of the light-harvesting complexes of photosystem I and II. The genes encoding LHC I and LHC II polypeptides are namedLhca1 throughLhca4 andLhcb1 throughLhcb6, respectively. The proposal follows the general format recommended by the Commision on Plant Gene Nomenclature. We also present a table for the conversion of old gene names to the new nomenclature.     

Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol

Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC), two metabolites of the environmental mutagen and carcinogen pentachlorophenol, were tested without exogenous activation in V79 Chinese hamster cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6-thioguanine resistance (TGr) and at the Na/K-ATPase locus to ouabain resistance (OuaR). Treatment was for 24 h at 37 degrees C. TCHQ produced statistically significant increases in the frequency of TGr mutants. The lowest observed effective dose (LOED) was 20 microM, where the relative cloning efficiency was 63%. The relationship between the dose of TCHQ and the frequency of TGr mutants was approximately linear over the range of 0-60 microM with an estimated slope (+/- 95% confidence limits) of 1.1 +/- 0.3 mutants per 10(6) clonable cells per microM. At the highest tested dose of TCHQ, 60 microM, the relative cloning efficiency was reduced to 7%. In contrast to TCHQ, TCC was unable to induce TGr mutants at doses up to 120 microM. The relative cloning efficiency at this dose was 5%. Both TCHQ and TCC were unable to induce OuaR mutants. The results suggest that TCHQ is at least partly responsible for the genotoxic activity of pentachlorophenol. TCHQ can produce reactive oxygen species, which may cause large genetic damage such as deletions, resulting in mutation to TGr but not to OuaR.     

Production of ψ-linolenic acid by the fungus Mucor rouxii on cheap nitrogen and carbon sources

Summary The production of -linolenic acid (GLA) by the fungus Mucor rouxii CBS 416.77 was studied on low budget nitrogen and carbon sources, i.e. rape meal, cocos expeller and two types of yeast extract (nitrogen sources), and starch, starch hydrolysate, beet molasses and cocos expeller (carbon sources). As references, Difco yeast extract and glucose were used. In flask cultivations the three yeast extracts were fully interchangeable, while the Difco yeast extract (the most expensive of those tested) gave a higher productivity of GLA in fermentor cultures (14 mg·l^–1·h^–1). The yield of lipids and GLA were increased in the order yeast extract < rape meal < cocos expeller. Thus the amount of lipid increased from 0.56 to 2.8 g·l^–1, and that of GLA from 0.15 to 0.33 g·l^–1. Use of beet molasses or cocos expeller as carbon sources gave poor growth. Starch and starch hydrolysate resulted in better productivity of GLA than glucose (4.7 and 4.9 compared to 3.4 mg·l^–1·h^–1). Offsprint requests to: A.-M. Lindberg     

Mathematical model for human myeloma relating growth kinetics and drug resistance

We present a computer-based mathematical model that can simulate characteristic features of the clinical time course of human myeloma. It asserts that therapy resistance in myeloma cells is an inherited trait associated with the longer inter-mitotic times of some cells and that the strength of this trait affects tumour growth characteristics. These kinetic differences within the malignant cell clone may also influence therapeutic efficacy. In the model, the same total therapy, administered in different time-dose fractions, could be 'curative' or 'minimally effective' depending on kinetic properties. For example, as others have shown, in myeloma pulsed intermittent therapy is often more effective than low dose continuous therapy. According to our model this finding is compatible with a high coefficient of inheritability of resistance from one cell generation to the next. The model also suggests that if there are subclones of varying resistance, a therapy must have some effect on each of them if it is to be employed in a curative fashion. While many aspects of the model are not yet clinically testable, exploration of its concepts might increase knowledge about fundamental neoplastic mechanisms.     

In vitro studies of biological effects of cigarette smoke condensate. II. Induction of sister-chromatid exchanges in human lymphocytes by weakly acidic, semivolatile constituents

Cigarette smoke condensate is known to enhance the frequency of sister-chromatid exchanges (SCE) in human lymphocytes in vitro and some of the activity has been found in the most volatile part of the particulate phase, the semivolatile fraction. In this study we have investigated the chemical composition and the SCE-inducing activity of the weakly acidic, semivolatile fraction of a cigarette smoke condensate. A number of individual weakly acidic compounds were also tested for their SCE-inducing effects. The weakly acidic fraction was separated by preparative gel chromatography into 11 subfractions (F1-F11). The chemical composition was determined by gas chromatography and gas chromatography-mass spectrometry. Measurements of the effects on SCE in human lymphocytes were used to evaluate the genotoxic effects. All fractions except F11 induced SCE in a dose-dependent way. The most active fraction was F4 which contained mainly alkyl-2-hydroxy-2-cyclopenten-1-ones. The individual compounds to be tested for induction of SCE were selected on the basis of their abundance in the weakly acidic subfractions and on the basis of their occurrence in the environment. Of 23 tested compounds, most of which were alkylphenols, 7 induced SCE, i.e., catechol, 2-(1-propenyl)phenol, cyclotene, maltol, isoeugenol, 2-methoxyphenol (guaiacol) and vanillin. Many of these are important flavor components that occur not only in tobacco and tobacco smoke but also in food, candies, beverages and perfumes.     

Purification and characterization of a new form (RLM2) of liver microsomal cytochrome P-450 from untreated rat

A new cytochrome P-450 isozyme (RLM2) has been purified to electrophoretic homogeneity from liver microsomes of the untreated rat. It has an apparent minimum molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 49,000. Absolute spectrum of the oxidized form indicates that this isozyme is essentially all in the low spin state. The maximum of the reduced CO complex is at 449 nm. Amino-terminal partial amino acid sequence and amino acid composition are different from those of RLM3 and RLM5, two other native forms of cytochrome P-450 previously reported from this laboratory as well as other forms reported in the literature. RLM2 is capable of oxidizing a variety of drug substrates, like benzphetamine and aminopyrine, and to a lesser extent ethoxycoumarin. With the steroid substrate multiple isomeric products are formed differentially. Progesterone is preferentially hydroxylated at the 15-position (15 beta-hydroxylation (34%) and 15 alpha-hydroxylation (13%) of the total) and at the 6 beta-position (21%). The major metabolite when testosterone was the substrate, 15 alpha-hydroxytestosterone, comprised 43% of the total, while a modest amount of 6 beta-hydroxytestosterone (12%) is formed. Another major metabolite (31%) has yet to be unequivocally identified, but is suggested to be 7 beta-hydroxytestosterone. Examination of the substrate dependence of major and minor isomeric metabolites provides evidence for a single substrate-binding site on RLM2. Regardless of the position hydroxylated, a common Km value was obtained. It is suggested that differences in formation of the isomeric and epimeric products relate to differences in distance from the active oxygen center and the position of attack.