Radiochemical detection of dihydrodiol dehydrogenase: distribution of the indomethacin sensitive enzyme in rat tissues
Dihydrodiol dehydrogenase catalyzes the NADP/sup +/ dependent oxidation of trans-dihydrodiols of polycyclic aromatic hydrocarbons (PAH) which are potent proximate carcinogens. The authors have developed a highly sensitive radiochemical assay for this enzyme in which the oxidation of trans-1,2-dihydroxy-3,5-cyclohexadiene, a model substrate for trans-dihydrodiol proximate carcinogens, is coupled to O-methylation catalyzed by catechol O-methyl transferase. Using S-adenosyl-(/sup 3/H-methyl)-methionine as methyl donor at a specific activity of 0.1 nCi/pmol and extracting the product, /sup 3/H-o-methoxyphenol, the assay provides a 5000 fold increase in sensitivity over the existing spectrophotometric method. The radiochemical assay was validated by comparing the K/sub m/ and V/sub max/ values for rat liver cytosol with those derived spectrophotometrically. In both instances there was close agreement between values (K/sub m/ = 0.77 +/- 0.11 mM and V/sub max/ = 2.14 +/- 0.13 nmoles/min/mg protein determined radiochemically; K/sub m/ = 0.96 +/- 0.10 mM and V/sub max/ = 6.31 +/- 0.50 nmoles/min/mg protein determined spectrophotometrically). Using the radiochemical method, dihydrodiol dehydrogenase activity was detected in the following rat tissues: liver > lung > heart > small intestine > testis > seminal vesicle > bladder > prostate > spleen. Specific activities ranged between 0.944 and 0.016 nmoles/min/mg protein. In liver, lung, and testis, which are sites of PAH metabolism, the dehydrogenase is sensitive to inhibition by low ..mu..M concentrations of indomethacin, suggesting that this drug can prevent the detoxification of proximate carcinogens by this route.
- Research Organization:
- Univ. of Pennsylvania, Philadelphia
- OSTI ID:
- 7022331
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
OXIDOREDUCTASES
RADIOASSAY
TISSUE DISTRIBUTION
POLYCYCLIC AROMATIC HYDROCARBONS
CARCINOGEN SCREENING
CARCINOGENS
LIVER
METABOLIC ACTIVATION
METHIONINE
RADIOCHEMISTRY
RATS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
VALIDATION
AMINO ACIDS
ANIMALS
AROMATICS
BODY
CARBOXYLIC ACIDS
CHEMISTRY
DIGESTIVE SYSTEM
DISTRIBUTION
DRUGS
ENZYMES
GLANDS
HYDROCARBONS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIPOTROPIC FACTORS
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
RODENTS
SCREENING
TESTING
VERTEBRATES
560300* - Chemicals Metabolism & Toxicology
550501 - Metabolism- Tracer Techniques