C.H. Woo

Alterations of specific forms of cytochrome P-450 in rat liver during acute carbon tetrachloride intoxication

Male rats were pretreated with phenobarbital, 3-methylcholanthrene, or the corresponding vehicles, administered carbon tetrachloride (CCl4), and sacrificed 3 hr later. Hepatic microsomes were isolated and assayed for cytochrome P-450 content and mixed-function oxidase activity. The residual content of cytochrome P-450 after CCl4 intoxication was similar in saline and phenobarbital-pretreated animals. Substantially greater amount of cytochrome P-450 remained in animals pretreated with 3-methylcholanthrene and then challenged with CCl4. In saline-pretreated animals, the dealkylation of 7-ethoxycoumarin, but not benzphetamine or N,N-dimethylaniline, was decreased following CCl4 exposure. The enhanced dealkylation following phenobarbital, but not following by 3-methylcholanthrene, was also decreased after CCl4 poisoning. Microsomes were solubilized with sodium cholate; cytochrome P-450 was partially purified by ω-octylamino Sepharose 4B column chromatography; and cytochrome P-450-containing elements were separated by SDS-polyacrylamide gel electrophoresis. A decrease in the staining intensity of a band migrating with a molecular weight of 51,600 was noted following CCl4 treatment. Furthermore, the phenobarbital-induced component (MW 51,600), but not that appearing after 3-methylcholanthrene induction (MW 57,900), was also diminished. Microsomal proteins separated by SDS-polyacrylamide gel electrophoresis and stained for heme also showed a decrease in staining which was greater in those microsomal proteins from phenobarbital and saline than in 3-methylcholanthrene-induced animals. The data suggest that a specific cytochrome P-450(s) in the saline-treated animals as well as the cytochrome P-450(s) induced by phenobarbital, but not the 3-methylcholanthrene-induced cytochrome P-450, are susceptible to CCl4-induced destruction.

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

Summary In intestinal mucosa of fed adult rats, cytochrome P-450 content and activity of benzpyrene hydroxylase and p -nitroanisole O-demethylase are highest in the upper small intestine and progressively decrease toward the terminal ileum. Among the mucosal cell populations, mature villous tip cells contain 6 to 10 times more cytochrome P-450 and drug-metabolizing activity per mg microsomal protein than epithelial crypt cells. On restriction of dietary iron intake for 48 hr, cytochrome P-450 content and drug-metabolizing enzyme activity of villous tip cells decrease to 42% and 13% of control values, but are restored within 24 hr by oral iron supplementation. These findings suggest that intestinal drug metabolism is localized primarily in upper villous cells of duodenal mucosa, that cytochrome P-450 is synthesized in maturing epithelial cells as they migrate to the top of the mucosal villus and that this process is critically dependent on dietary (luminal) iron.

Cellular distribution of cytochrome P-450 loss in rats of different ages treated with alkyl halides

Abstract Challenge of male rats with a single dose of the alkyl halides, 1,2-dibromo-3-chloropropane (DBCP), ethylene dibromide (EDB), carbon tetrachloride (CCl 4 ), and epichlorohydrin (EPI), resulted in significant decreases in cytochrome P -450 in microsomes isolated from liver, kidney, testis, lung, and small intestinal mucosa 48 hr after treatment. Treatment with CCl 4 , but not DBCP, EDB, or EPI, was characterized by rapid loss of cytochrome P -450, detectable within 4 hr. Evidence of lipid peroxidation was found only in hepatic microsomes from rats treated with CCl 4 , but not in hepatic or extrahepatic microsomes from rats treated with other compounds. In liver tissue, treatment with DBCP and CCl 4 resulted in a decrease in cytochrome P -450 in both rough and smooth microsomal fractions and nuclei, but not in mitochondrial fractions. Mixed-function oxidase (MFO) activities in hepatic microsomes decreased in parallel with cytochrome P -450 content after treatment with DBCP and EDB. In microsomes and nuclei after treatment with CCl 4 and in nuclei after treatment with DBCP, however, the response of the MFO depended on the substrate tested. Microsomal cytochrome P -450, which is susceptible to proteolytic cleavage, and microsomal and nuclear cytochrome P -450, which increased with maturation and decreased with aging of the rat, appeared to be the most responsive of the forms of cytochrome P -450 to alkyl halide treatment. These results suggest that treatment with alkyl halides may preferentially affect specific isozymes of cytochrome P -450.

In vitro release of α1-acid glycoprotein RNA sequences shows fidelity with the acute phase response in vivo

The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in α1-acid glycoprotein (αAGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron αAGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, αAGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A) + RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some αAGP sequences in smaller, more heterogeneous poly(A)-RNA, and leakage of some αAGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature αAGP mRNA to the nucleus. In contrast to αAGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest αAGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A) + RNA preparations which may represent 3′-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of αAGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of αAGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of αAGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of αAGP in the acute phase response.

Independent responses of nucleoside triphosphatase and protein kinase activities in nuclear envelope following thioacetamide treatment

Abstract Two methods were used to prepare rat-liver nuclear envelope from control and thioacetamide-treated animals. The method employing DNase-digestion and Tris-EDTA dissociation of ribosomes resulted in much lower specific NTPase and protein kinase activities. Monnerons preparative technique yielded NE preparations with high specific activities. Following thioacetamide treatment, the NE NTPase increased to nearly 300% of the control activity, with no change in the protein kinase activity. It appears that the NE protein kinase activity is not related to NE NTPase and is not involved in RNA transport.

Modulation of RNA transport by polyvinylpyrrolidone

A number of studies have documented substantial loss of nuclear protein during aqueous nuclear isolation procedures. This loss can, to some extent, be counteracted by addition of impermeable macromolecules like polyvinylpyrrolidone, which prevent nuclear swelling. While nucleic acids appear to be much less susceptible to leakage during isolation, the effects of these additives on RNA release duringin vitro incubation have not been examined. Here we show that addition of polyvinylpyrrolidone results in significant decreases in RNA transport; inhibition becomes maximal at 50–75 μM addition.

Polypeptide composition of nuclear envelope following thioacetamide-induced nuclear swelling

Abstract Nuclear envelopes (NE) 1 were isolated from rat liver following thioacetamide-induced nuclear swelling. At 8 and 48 h intervals following thioacetamide treatment, periods which correspond to nuclear swelling phases, NE showed little change in polypeptide composition (although the polypeptide composition depended to some extent upon the isolation procedure used). Since the amount of NE protein increases by 30–40% and 70–80% at these times (respectively), the data suggest that concerted synthesis of a number of NE polypeptides occurred. We propose that triggered expansion of the NE may be an important first stage in the action of carcinogens.