Surendra K. Bansal

Glucuronidation of oxygenated benzo(a)pyrene derivatives by UDP-glucuronyltransferase of nuclear envelope

Abstract Several benzo(a)pyrene phenols, dihydrodiols, epoxides, quinones, and a diolepoxide were tested as possible substrates for UDP-glucuronyltransferase located in rat liver nuclear envelope fraction. Only phenolic derivatives of benzo(a)pyrene served as substrates for the nuclear membrane transferase, under the conditions tested. The specific activities observed in nuclear envelope preparations were greater than or equal to, those of the microsomal fraction indicating that a very effective detoxication mechanism for these phenols is present in the nuclear compartment.

High pressure liquid chromatographic separation of multiple forms of cytochrome P-450

The major form of cytochrome P-450 isolated and purified from the hepatic microsomes of phenobarbital pretreated rats by sequential chromatography on n-octylamino-Sepharose 4B and DEAE-cellulose columns was found to be homogeneous by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. However, this cytochrome P-450 was resolved into three bands by high pressure liquid chromatography on an Anpac ion-exchange column. High pressure liquid chromatography isolated forms had similar molecular weights of 55,000 with lambda max of the CO-reduced difference spectrum at 450 nm and were found to be in the low spin state. The results demonstrate the effectiveness of high pressure liquid chromatography in the resolution of cytochrome P-450s of similar molecular weights but different net charges.

Resolution of multiple forms of cytochrome P-450 by high-performance liquid chromatography

The major forms of cytochrome P-450 in the hepatic microsomes of rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3MC) were isolated by sequential chromatography on n-octylamino-Sepharose 4B and DEAE-cellulose columns. These preparations exhibited single protein bands corresponding to cytochrome P-450s by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). High-performance liquid chromatography (HPLC) of these preparations on an anion-exchange column yielded three peaks from the PB-induced major cytochrome P-450 and a single peak from the 3MC-induced major cytochrome P-450. That the HPLC-isolated protein peaks were various forms of cytochrome P-450 was confirmed by spectral examination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Examination of their absolute spectra revealed these cytochrome P-450s to be in a low-spin state. The lambda max of the reduced CO-complex spectra and molecular weights were found to be 450 nm and 53,000, respectively, for all the three HPLC-resolved cytochrome P-450s from PB-induced rats; and 448 nm and 56,000, respectively, for the HPLC-isolated cytochrome P-450 from 3MC-induced rats. The results demonstrate the effectiveness of HPLC in the determination of cytochrome P-450 multiplicity and charge heterogeneity.

Aflatoxin B1-4-hydroxylase is associated with cytochrome P3-450 in C57BL6 mouse liver

Messenger RNA from the livers of Aroclor 1254 treated mice was used to produce a cDNA library. cDNA clones corresponding to cytochromes P1-450 and P3-450 were isolated from this library by screening with a probe for the rat cytochrome P-450c gene. Specific non-cross hybridizing probes for P1-450 and P3-450 were prepared from unique restriction fragments. The radiolabeled probes were hybridized to RNA from mice treated with a low (15 mg/kg) and high (150 mg/kg, 400 mg/kg) doses of beta-naphthoflavone. The low dose of beta-naphthoflavone was found to induce only P3-450 mRNA, whereas higher doses induced both P1-450 and P3-450 mRNA. Similarly, a low dose of beta-naphthoflavone induced aflatoxin B1-4-hydroxylase, whereas higher doses induced both aflatoxin B1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. These results suggest that P3-450 mRNA codes for the cytochrome that is associated with aflatoxin B1-4-hydroxylase activity.