No abstract

Bacterial lipopolysaccharide (LPS) and a diverse array of other immunostimulants and cytokines suppress the metaboism of endogenous and exogenous substances by reducing activity of the hepatic cytochrome P450 mixedfunction oxidase system. Although this effect of immunostimulants was first described almost 40 yr ago, the mechanism is obscure. Immunostimulants are now known to cause NO overproduction by celis via induction of nitric oxide synthase.We have investigated whether NO overproduction is involved in suppressing hepatic metabolism by LPS. In vitro treatment of hepatic microsomes with NO, produced by chemical decomposition of 3-morpholinosydnonimnue or by nitric oxide synthase, substantially suppressed cytochrome P450-dependent oxygenation reactions. This effect of NO was seen with hepatic microsomes prepared from two species (rat and chicken) and after exposure to chemicais that induce distinct molecular isoforms of cytochromes P450 (-naphthoflavone, 3-methylcholanthrene, and phenobarbital). Spectral studies indicate that NO reacts in vitro with both Fe2+-and Fe3+-hemes in microsomal cytochromes P450. In vivo, LPS diminished the phenobarbital-induced dealkylation of 7-pentoxyresorufin by rat liver microsomes and reduced the apparent P450 content as measured by CO binding. These LPS effects were associated with induction of NO synthesis; LPS-induced NO synthesis showed a strong positive correlation with the severity of cytochrome P450 inhibition. The decrease in both hepatic microsomal P450 activity and CO binding caused by LPS was largely prevented by the selective NO synthase inhibitor N"-nitro-L-arginine methyl ester. Our rmdings implicate NO overproduction as a major factor mediating the suppression of hepatic metabolism by immunostimulants such as LPS.NO is a secretory product of mammalian cells that is important in the regulation of vascular tone, platelet function, neurotransmission, and host-defense mechanisms (1-3). These physiological actions are attributable to the oxidation by NO of heme and nonheme iron and iron-sulfur complexes in the active sites of key metabolic enzymes. Thus, through its interaction with iron, NO modulates the activity of target proteins. Although NO is normally produced in relatively small quantities and presumably by particular cell types (e.g., endothelial cells and neurons), NO (PB) (CYP2B1/2), (-naphthoflavone (3-NF), and 3-methylcholanthrene (3-MC) (CYPlA1/2) (4-7).Immunological stimuli depress cytochrome P450-mediated hepatic metabolism of a variety of drugs and endogenous substances. Indeed, attenuated cytochrome P450 activity has been seen in animals after infection with bacteria and viruses or after treatment with cytokines (e.g., interleukin 1; interferon 'y, tumor necrosis factor a) and immunostimulants (e.g., LPS and single-stranded RNA) (8, 9). Despite wide acceptance that the immune system can inhibit hepatic drug metabolism, the mechanism of this effect is largely unknown. Our study reveals that NO, produced by immunoactivated cells, mediates sup...

The environmental toxin TCDD (2,3,7,8-tetrachlorodibenzop-dioxin, dioxin) produces diverse toxic effects including a lethal wasting syndrome whose hallmark is suppressed hepatic gluconeogenesis. All TCDD toxicities require activation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor. Whereas the mechanism for AHR induction of target genes is well understood, it is not known how AHR activation produces any TCDD toxicity. This report identifies for the first time an AHR target gene, TiPARP (TCDD-inducible poly(ADPribose) polymerase, PARP7) that can mediate a TCDD toxicity, i.e. suppression of hepatic gluconeogenesis. TCDD suppressed hepatic glucose production, expression of key gluconeogenic genes, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase), and NAD ؉ levels, and increased PARP activity and

Photooxidized tryptophan (TRP) in tissue culture medium elicits a transient cytochrome P450 (CYP1) induction response in cultured cells. We show here that exposure of TRP to window sunlight (aTRP) greatly increased the potency, efficacy, and duration of CYP1A induction by TRP in primary chick embryo hepatocytes and in vivo. Aqueous TRP exposed to sunlight for 7 days exhibited a 100-fold or greater increase in potency over TRP in medium. The induction response was sustained for at least 48 h and was comparable in efficacy to 2,3,7,8-tetrachlorodibenzo-p-dioxin. In hepatocytes, increases in mRNAs for CYP1A4 and CYP1A5, chick orthologs of mammalian CYP1A1 and 1A2, preceded increases in CYP1A proteins and enzyme activities, 7-ethoxyresorufin deethylase (EROD) for CYP1A4 and arachidonic acid epoxygenation for CYP1A5, consistent with a transcriptional mechanism. Aryl hydrocarbon receptor (AhR) dependence was evidenced by aTRP induction of EROD in wild-type Hepa1c1c7 cells but not in AhR-defective (c35) mutants. Preparations of aTRP were stable for many months at 4 degrees C and were relatively resistant to metabolism by hepatocytes or liver microsomes. Fractionation of aTRP by HPLC analysis coupled with EROD assays showed that aTRP contained multiple photoproducts and CYP1A inducing components, which varied in sensitivity to metabolism by hepatocytes. The previously identified TRP photoproduct, 6-formylindolo[3,2-b]carbazole (FICZ), was one component, but FICZ was not required for CYP1A induction by the aTRP mixture. These findings identify the indoor environment, and window sunlight in particular, as a new source of CYP1A inducers. Further, the evidence that biologically active metabolites of an endogenous substrate, arachidonic acid, are formed by aTRP-induced CYP1A provides a pathway by which TRP photoproducts, like toxic xenobiotics, could have significant physiologic effects.

No abstract

Toxicologic and physiologic roles of CYP1A enzyme induction, the major biochemical effect of aryl hydrocarbon receptor activation by TCDD and other receptor ligands, are unknown. Evidence is presented that CYP1A exerts biologic effects via metabolism of endogenous substrates (i.e., arachidonic acid, other eicosanoids, estrogens, bilirubin, and melatonin), production of reactive oxygen, and effects on K(+) and Ca(2+) channels. These interrelated pathways may connect CYP1A induction to TCDD toxicities, including cardiotoxicity, vascular dysfunction, and wasting. They may also underlie homeostatic roles for CYP1A, especially when transiently induced by common chemical exposures and environmental conditions (i.e., tryptophan photoproducts, dietary indoles, and changes in oxygen tension).

Transcriptional regulation by the aryl hydrocarbon receptor, for which the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent ligand, leads in mammalian liver to the induction of genes for two distinct cytochrome P450 (CYP)1A enzymes, CYP1A1 and -1A2. Fish seem to have only one CYP1A enzyme. CYP1A enzymes have been regarded as injurious largely because of their ability to activate chemical carcinogens. We report here the cloning and sequencing of cDNAs for two catalytically distinct TCDD-induced CYP enzymes in chick embryo liver. One mediates classic CYP1A1 activities. The other has some -1A2-like activities and is also responsible for TCDD-induced arachidonic acid epoxygenation, a much more conspicuous effect in liver of chicks than of mammalian species. Amino acid sequence analysis shows that although each chick enzyme can be classified in the CYP1A family, both are more like CYP1A1 than -1A2, and neither can be said to be directly orthologous to CYP1A1 or -1A2. Phylogenetic analysis shows that the two chick enzymes form a separate branch in the CYP1A family tree distinct from mammalian CYP1A1 and -1A2 and from fish CYP1A enzymes. The findings suggest that CYP1A progenitors split into two CYP enzymes with some parallel functions independently in two evolutionary lines, evidence for convergent evolution in the CYP1A family. Northern analysis shows that the chick enzymes have a different tissue distribution from CYP1A1 and -1A2. Polymerase chain reaction and in situ hybridization data show that both chick enzymes are expressed in response to TCDD even before organ morphogenesis. The findings further suggest that beyond their role in activating carcinogens, CYP1A enzymes have conferred evolutionary and developmental advantages, perhaps as defenses in maintaining homeostatic responses to toxic chemicals. Activation of the aryl hydrocarbon (Ah)1 receptor by toxic polyhalogenated aromatic hydrocarbons like TCDD leads to the transcriptional induction of genes in the CYP1 family and to a toxicity syndrome that includes immunologic, hormonal, and cardiac dysfunction and tumor promotion (1, 2). There are large unexplained species differences in sensitivity to TCDD toxicity that cannot be attributed to differences in Ah receptor concentration or binding affinity and seem to involve factors downstream of the Ah receptor (1, 2). Ligand activation of the Ah receptor in mammalian liver induces expression of CYP1A1 and -1A2 and the more recently discovered CYP1B1 (3). These enzymes exhibit differences in substrate specificity, regulation, and tissue distribution. Because single amino acid differences can change CYP catalytic activity (4), species differences in CYP1 structure and function may contribute to differences in sensitivity to TCDD toxicity. The chicken and chick embryo close to hatching are among the more sensitive species to TCDD toxicity. They have long served as models for the study of Ah receptor-mediated toxicity and changes in heme and hemoprotein synthesis (2), but the chick TCD...

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver . Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell . The final fraction contains 5'-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes . In the presence of 0 .01 M Mg++, 5'-nucleotidase exhibits a double pH optimum at pH values of 7 .5 and 9 .5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts . Cytochrome P-450 is not detectable . Na+-K+-activation of ATPase is observed to the extent of 20-36 0/, in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi .