Håkan Wallin

K-ras mutations in sinonasal adenocarcinomas in patients occupationally exposed to wood or leather dust

Of 39 males diagnosed with sinonasal adenocarcinomas over 30 years in the Lund University Hospital catchment area (1.5 million inhabitants), archival tumor tissue was available from 29. Of these, 16 had been exposed to wood dust and three had been exposed to leather dust. The intestinal-type and papillary adenocarcinomas were more common in the exposed patients (P = 0.0002, Fishers exact test). The tumors from all but one of the 29 sinonasal adenocarcinomas could be analyzed for point mutations at codons 12, 13 and 61 of the K-ras gene. Four mutations were detected in the 28 tumors. The three mutations in the patients exposed to wood and leather dust were all G:C --> A:T transitions, with two at position 2 of codon 12 and one at position 2 of codon 13. The high proportion of G:C --> A:T mutations in this rare tumor may reflect a genotoxic agent in wood and leather dust.

A rapid and sensitive method for determination of covalent binding of benzo[a]pyrene to proteins

A method is presented for the quantitative determination of covalent binding of metabolically activated benzo[a]pyrene to microsomal proteins. After incubation of radiolabelled benzo[a]pyrene with microsomes and NADPH, the mixture is applied to filter paper discs. These are immersed in ethanol to precipitate the proteins. Unbound radiolabel is removed by repeated washes of the filters in organic solvents before scintillation counting. The method is simple, rapid, sensitive and accurate, and works both with 14C- and 3H-labelled compounds. The method is suitable for measuring the incorporation of other radiolabelled xenobiotics to proteins of both microsomes and other subcellular fractions and for the analysis of binding to isolated proteins.

Activation of microsomal glutathione transferase activity by reactive intermediates formed during the metabolism of phenol

The activity of microsomal glutathione transferase was increased 1.7-fold in rat liver microsomes which carried out NADPH dependent metabolism of phenol. Known phenol metabolites were therefore tested for their ability to activate the microsomal glutathione transferase. The phenol metabolites benzoquinone and 1,2,4-benzenetriol both activated the glutathione transferase in microsomes 2-fold independently of added NADPH. However, NADPH was required to activate the enzyme in the presence of hydroquinone. Catechol did not activate the enzyme in microsomes. The purified enzyme was activated 6-fold and 8-fold by 5 mM benzenetriol and benzoquinone respectively. Phenol, catechol or hydroquinone had no effect on the purified enzyme. When microsomal proteins that had metabolized [14C]phenol were examined by SDS polyacrylamide gel electrophoresis and fluorography it was found that metabolites had bound covalently to a protein which comigrated with the microsomal glutathione transferase enzyme. We therefore suggest that reactive metabolites of phenol activate the enzyme by covalent modification. It is discussed whether the binding and activation has general implications in the regulation of microsomal glutathione transferase and, since some reactive metabolites might be substrates for the enzyme, their elimination through conjugation.

Evidence that covalent binding of metabolically activated phenol to microsomal proteins is caused by oxidised products of hydroquinone and catechol.

The metabolic activation of [14C]phenol resulting in covalent binding to proteins has been studied in rat liver microsomes. The covalent binding was dependent on microsomal enzymes and NADPH and showed saturation kinetics for phenol with a Km-value of 0.04 mM. The metabolites hydroquinone and catechol were formed at rates which were 10 or 0.7 times that of the binding rate of metabolically activated phenol. The effects of cytochrome P-450 inhibitors and cytochrome P-450 inducers on the metabolism and binding of phenol to microsomal proteins, suggest that cytochrome P-450 isoenzyme(s) other than P-450 PB-B or P-450 beta NF-B catalyses the metabolic activation of phenol. Furthermore, reconstituted mixed-function oxidase systems containing cytochrome P-450 PB-B and P-450 beta NF-B were (on basis of cytochrome P-450 content) 6 and 11 times less active in catalysing the formation of hydroquinone than microsomes. The isolated metabolites hydroquinone and catechol bound more extensively to microsomal proteins than phenol and the binding of these was not stimulated by NADPH. The binding occurring during the metabolism of phenol could be predicted by the rates of formation of hydroquinone and catechol and the rates by which the isolated metabolites were bound to proteins.

Enzyme immunoassay of benzo[a]pyrene conjugated to DNA, RNA and microsomal proteins using a monoclonal antibody

An enzyme immunoassay for the detection of benzo[a]pyrene covalently conjugated to macromolecules has been developed. The monoclonal antibody, raised through in vitro immunization reacted with benzo[a]pyrene metabolites bound to DNA, RNA and proteins. The lower detection limit for the assay was 1 pmol for benzo[a]pyrene bound to DNA or RNA, and 5 pmol when bound to protein.

Covalent binding of benzo[a]pyrene to cytochrome P-450βNF-B2 and other proteins in reconstituted mixed-function oxidase systems

A reconstituted mixed-function oxidase system, containing the major beta-naphthoflavone-induced isozyme of rat liver cytochrome P-450 bound benzo[a]pyrene covalently in the presence of NADPH. NADPH-cytochrome P-450 reductase was required for binding and a maximum rate of adduct formation was obtained at 8 units of reductase per nmol cytochrome P-450. Phosphatidylcholine inhibited this reaction. Benzo[a]pyrene was bound to the cytochrome, but not to the reductase, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Approximately 6 molecules of benzo[a]pyrene bound to each molecule cytochrome P-450 during prolonged incubations. No binding occurred when the beta-naphthoflavone-induced isozyme of cytochrome P-450 was replaced by the major isozyme induced by phenobarbital, but both cytochromes incorporated benzo[a]pyrene to approximately the same extent when they were incubated together in the presence of the reductase and NADPH. Metabolically activated benzo[a]pyrene also bound covalently to purified epoxide hydrolase, when this enzyme was added to the reconstituted mixed-function oxidase system.