Robert J. Highet

Nephrotoxicity of chloroform: Metabolism to phosgene by the mouse kidney

In this investigation, we have attempted to determine whether chloroform (CHCl3)-induced nephrotoxicity might be due to its metabolism to phosgene (COCl2) in the kidney. We have found that kidney homogenates from DBA/2J male mice in the presence of glutathione metabolize CHCl3 to 2-oxothiazolidine-4-carboxylic acid (OTZ). This product appears to be formed by the initial trapping of COCl2 by two molecules of GSH to form diglutathionyl dithiocarbonate (GSCOSG). Kidney gamma-glutamyl transpeptidase can rapidly metabolize GSCOSG to N-(2-oxothiazolidine-4-carbonyl)-glycine which is then hydrolyzed, possibly by cysteinyl glycinase to OTZ. The finding that deuterium-labeled chloroform (CDCl3) was less nephrotoxic and depleted less renal GSH than did CHCl3 suggests that the metabolism of CHCl3 to COCl2 may also occur in the kidney in vivo and lead to nephrotoxicity.

(5Z,9Z)-3-alkyl-5-methylindolizidines fromSolenopsis (Diplorhoptrum) species

The alkaloidal venom components of two species of thief ants,Solenopsis (Diplorhoptrum) species AA andS. (Diplorhoptrum)conjurata have been found to contain (5Z,9Z)-3-hexyl-5-methylindolizidine and a mixture of (5Z,9Z)-3-ethyl-5-methylindolizidine andcis-2-methyl-6-nonyl-piperidine,trans-2-methyl-6-nonylpiperidine,cis-2-methyl-6-undecylpiperidine, and hexadecanoic acid.Monomorium pharaonis was similarly investigated and found to contain the indolizidine and pyrrolidines previously described (Ritter et al., 1977b). Both indolizidines were synthesized along with their stereoisomers and separated by preparative gas chromatography. Spectral studies revealed the stereochemistry to be 5Z,9Z in both cases. The stereochemistry of 2-butyl-5-pentylpyrrolidine inM. phaeronis has also been established. Biosynthetic relationships are discussed.

Stereoselective formation of bromobenzene glutathione conjugates.

Two bromobenzene-glutathione conjugates have been detected as both in vivo and in vitro metabolites of bromobenzene. Separation and purification by high pressure liquid chromatography (HPLC) and analysis by 13C and 1H-NMR spectroscopy indicated that the metabolites are trans-3-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol and trans-4-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol. The two conjugates are formed in unequal amounts; over a dose range of 25-500 mg/kg the ratio of the two conjugates excreted into bile in 6 h was 1.6 +/- 0.1 (mean +/- S.E.). Pretreatment of rats with either phenobarbital or 3-methyl-cholanthrene did not significantly alter the ratio of the two conjugates excreted into bile. When bromobenzene was incubated with rat liver microsomes and glutathione, the same two conjugates were formed in the presence but not in the absence of 100 000 x g supernatant. Furthermore, in the presence of 100 000 x g supernatant from control animals, microsomes from rats treated with phenobarbital formed both conjugates 6 times more rapidly than did microsomes from control rats, whereas microsomes from rats treated with 3-methylcholanthrene formed both conjugates less rapidly than did those from control rats. Thus, the data suggest that both conjugates are formed via bromobenzene 3,4-oxide and that their formation requires in liver cytosol.

Differences in the localization and extent of the renal proximal tubular necrosis caused by mercapturic acid and glutathione conjugates of 1,4-naphthoquinone and menadione☆

We have previously demonstrated that administration of various benzoquinol-glutathione (GSH) conjugates to rats causes renal proximal tubular necrosis and the initial lesion appears to lie within that portion of the S3 segment within the outer stripe of the outer medulla (OSOM). The toxicity may be a consequence of oxidation of the quinol conjugate to the quinone followed by covalent binding to tissue macromolecules. We have therefore synthesized the GSH and N-acetylcysteine conjugates of 2-methyl-1,4-naphthoquinone (menadione) and 1,4-naphthoquinone. The resulting conjugates have certain similarities to the benzoquinol-GSH conjugates, but the main difference is that reaction with the thiol yields a conjugate which remains in the quinone form. 2-Methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone caused a dose-dependent (50-200 mumol/kg) necrosis of the proximal tubular epithelium. The lesion involved the terminal portion of the S2 segment and the S3 segment within the medullary ray. At the lower doses, that portion of the S3 segment in the outer stripe of the outer medulla displayed no evidence of necrosis. In contrast, 2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) caused no apparent histological alterations to the kidney. 2-(Glutathion-S-yl)-1,4-naphthoquinone and 2,3-(diglutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) were relatively weak proximal tubular toxicants and the lesion involved the S3 segment at the junction of the medullary ray and the OSOM. A possible reason(s) for the striking difference in the toxicity of the N-acetylcysteine conjugate of menadione, as opposed to the lack of toxicity of the GSH conjugate of menadione, is discussed. The basis for the localization of the lesion caused by 2-methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone requires further study.

Systematic implications of the exocrine chemistry of some Hypoclinea species

Abstract The exocrine compounds produced by several species of Hypoclinea were analysed and compared to those identified in species in other genera of the ant subfamily Dolichoderinae. Two new natural products, 2-hydroxy-6-methylaceto-phenone and 2-acetoxy-6-methylacetophenone, were identified as anal gland products of three Hypoclinea species. The significance of two “forms” of H. bidens possessing completely different glandular secretions is discussed, and the relationship of the genera Hypoclinea and Dolichoderus is explored in terms of the exocrine chemistry of species in these two dolichoderine taxa.

Carbon-13 nuclear magnetic resonance studies of spironolactone and several related steroids

The 13C chemical shifts for all the carbon atoms is spironolactone have been assigned. Assignments for nine additional steroids which include the C-7 beta isomer of spironolactone, its C-7 thiol hydrolysis product, the 7 alpha-thioacetate derivative of testosterone and its thiol hydrolysis product are also reported.

The use of stable isotopes to identify reactive metabolites and target macromolecules associated with toxicities of halogenated hydrocarbon compounds.

1. Halogenated compounds, such as the inhalation anaesthetics, halothane and enflurane, and the chemicals chloroform, carbon tetrachloride, and bromotrichloromethane can cause hepatotoxicity, nephrotoxicity, and inactivation of cytochromes P-450. Each of these toxicities is mediated by reactive metabolites. 2. Stable isotopes of hydrogen, carbon, chlorine and oxygen have been used in conjunction with mass spectrometry and n.m.r. spectrometry to identify the structures of these metabolites, to elucidate the mechanisms of their formation, and to characterize the structures of their macromolecular adducts. 3. In a number of cases, oxidative pathways of metabolism to toxic metabolites have been defined by kinetic deuterium isotope effects. 4. Recently, we have found that the trichloromethyl radical metabolite of bromotrichloromethane can activate myoglobin by causing the covalent cross-linking of haem to protein. The structure of a haem-myoglobin adduct has been defined by the use of stable isotope studies.

Microbial hydroxylation of a dihydroartemisinin derivative

The fungus Beauveria sulfurescens has been employed to convert the 14-methyl group of the phenylcarbamoyl of dihydroartemisinin to the corresponding 14-hydroxymethyl derivative; the structure of the product was established by mass and 2D NMR spectroscopy.