P-450 Mixed-function Oxidase Activities Research Articles

Polybrominated biphenyl induction of cytochrome P450 mixed function oxidase activity in primary rat and human hepatocytes

Polybrominated biphenyl (PBB) is an industrial chemical and environmental contaminant with known incidence of significant human exposure. PBB has been studied in laboratory animals and found to have significant toxicological effects as well as being a potent inducer of hepatic cytochrome P450 mixed function oxidase (MFO) activity. As part of our program to compare the response of laboratory animals and humans to industrial and environmental toxicants, we studied the effect of a major component of commercial PBB mixtures, 2,2′,4,4′,5,5′-hexabromobiphenyl (HBB), on MFO induction in primary cultures of human and rat hepatocytes. MFO induction was evaluated by measuring the deethylation of 7-ethoxycoumarin by intact hepatocytes. Rat hepatocytes were found to be highly susceptible to HBB induction of 7-ethoxycoumarin O-deethylase (ECOD) activity, with significant induction observed at the lowest concentration tested of 10 −8 M. Human hepatocytes were found to have a higher threshold for HBB induction of ECOD activity than rat hepatocytes. The lowest concentration of HBB required for ECOD induction observed for human hepatocytes was 10- to 1000-fold higher (10 −7, 10 −6, 10 −5 and 10 −5 M for the four human samples) than that found in rat hepatocytes. Future mechanistic investigation of this observed difference in sensitivity towards PBB between rat and human hepatocytes may aid the extrapolation of human health risk from toxicological data obtained from laboratory animals.

Effects of m-xylene on rat nasal cytochrome P450 mixed function oxidase activities

The effect of m-xylene on the rat nasal cytochrome P450 (P450) mixed function oxidase system was analyzed in vitro utilizing microsomes isolated 2, 12, and 24 h following intraperitoneal administration of this solvent in vivo. For comparative purposes, pulmonary and hepatic activities were also measured. Benzyloxyresorufin O-deethylation (BROD) and ethoxyresorufin O-deethylation (EROD), catalytic activities linked with P450 isozymes IIB1 and IA1, respectively, were inhibited in nasal tissue at all times following m-xylene administration. Pulmonary tissue mimicked this m-xylene-dependent inhibition of BROD activity but did not display significant inhibition of EROD activity. In contrast, m-xylene caused a dramatic induction of both BROD and EROD activity in hepatic tissue. The metabolism of a third P450 substrate, cyclopentadienyl manganese tricarbonyl (CMT), was also analyzed. m-Xylene caused significant inhibition of CMT metabolism at all time points in both nasal and pulmonary microsomes but was without effect on hepatic microsomal metabolism of this compound. These data show an inhibitory effect of m-xylene on rat nasal and pulmonary but not hepatic cytochrome P450-dependent metabolism.

Characterization of a microsomal fraction from rat small intestine for metabolic activation of some promutagens.

Rat small intestine possesses cytochrome P-450 mixed function oxidase activity and is thus potentially capable of activating procarcinogens during absorption. The low spontaneous tumour incidence at this site may be due partially to the detoxification activity of the intestinal mucosa. To study the contribution of the intestine to the metabolism of foreign chemicals, microsomes have been obtained from rat small intestine by a procedure facilitating recovery of preparations with consistently high enzyme activities and abilities to activate some selected promutagens in the Salmonella mutagenicity assay. Intestinal microsomes from animals with and without pretreatment with inducers have been used for investigations of biochemical properties and ability to activate several mutagens in the Salmonella plate incorporation assay. The effects of microsomal protein concentration and inhibitors were also studied. The results are compared with data obtained using liver microsomes from the same animals. Despite the induction of lower numbers of revertants, intestinal microsomes were at least as efficient as liver preparations for the activation of all the promutagens used when the data were corrected for cytochrome P-450 contents. Differences in dose-response curves for certain mutagens using liver and intestinal microsomes are discussed in relation to variation in metabolism of promutagens. Activation was linearly dependent on microsomal protein concentration, for both liver and small intestinal microsomes. The intestine was generally less susceptible to the effects of cytochrome P-450 and P-448 inducers, although sensitivity to orally administered phenobarbitone was increased by extending treatment times. SKF525A and beta-naphthoflavone inhibited microsomes from both sources, equal inhibition being observed for each type following incorporation of the inhibitor, although they differed in their ability to activate 2-acetylaminofluorene in the presence of the deacylase inhibitor, NaF. The data are discussed in relation to the possible role of the small intestine in metabolic activation in vivo and the utility of microsomes therefrom for the in vitro detection of putative dietary carcinogens.