Hepatic P450 activities are profoundly affected by various infectious and inflammatory stimuli, and this has clinical and toxicological consequences. Whereas the expression of most P450s in the liver is suppressed, some are induced. Many of the effects observed in vivo can be mimicked by pro-inflammatory cytokines and IFNs, and P450s are differentially regulated by these agents. Therefore, different cytokine profiles and concentrations in the vicinity of the hepatocyte in different models of inflammation may result in qualitatively and quantitatively different effects on populations of P450s. In addition to cytokines, glucocorticoids may have an important role in P450 regulation in stress conditions, including that caused by inflammatory stimuli. Although in many cases the decreases in activity are due primarily to a down-regulation of P450 gene transcription, it is likely that modulation of RNA and protein turnover, as well as enzyme inhibition, contributes to some of the observed effects. The mechanisms whereby these effects are produced may also vary with both the P450 under study and the time course of the effect. The complexity of the P450 response to inflammation and infection means that all of the above factors must be considered when trying to predict the effect of a given infectious or inflammatory condition on the clinical or toxic response of humans or animals to an administered drug or toxin. The question of whether the down-regulation of the hepatic P450 system to inflammation or infection is a homeostatic or pathological response cannot be answered at present. It is difficult to discern the physiological benefit of reducing hepatic P450 activities, unless it is to prevent the generation of reactive oxygen species generated by uncoupled catalytic turnover of the enzymes. On the other hand, as we proposed some years ago [64], the suppression of P450 may be due to the liver's need to utilize its transcriptional machinery and energy for the synthesis of APPs involved in the inflammatory response. In that case, one could ask why the organism has gone to the trouble of employing differential mechanisms for suppression of P450. One answer could be that the response evolved after the divergence of many of the P450 genes, necessitating the evolution of multiple redundant mechanisms for P450 suppression. In contrast to the down-regulation of P450s in the liver, the induction of several forms in this and other tissues suggests a more specific homeostatic role of these effects, e.g., in generation or catabolism of bioactive metabolites.

Regulation of Cytochromes P450 During Inflammation and Infection

(1981). Inhibitors of Cytochrome P-450s and Their Mechanism of Action. Drug Metabolism Reviews: Vol. 12, No. 1, pp. 1-117.

Inhibitors of Cytochrome P-450s and their mechanism of action.

A method for isolation of mouse liver cells by a two-step perfusion with calcium and magnesium-free Hanks' salt solution followed by a medium containing collagenase is described. Several variations of the commonly used procedure for rat liver cell isolation were quantitatively compared with respect to cell yield and viability. The optimal isolation technique involved perfusion through the hepatic portal vein and routinely produced an average of 2.3 x 10(6) viable liver cells/g body weight. Optimal perfusate collagenase concentration was found to be 100 U of enzyme activity per milliliter of perfusate. Light and electron microscopic evaluation of liver morphology after several steps of the isolation showed distinct morphologic changes in hepatocytes and other liver cells during perfusion. After perfusion with Hanks' calcium- and magnesium-free solution, many hepatocytes exhibited early reversible cell injury. These changes included vesiculation and slight swelling of the endoplasmic reticulum as well as mitochondrial matrix condensation. Subsequent to perfusion with collagenase, the majority of hepatocytes appeared connected to one another only by tight junctional complexes at the bile canaliculi. Multiple evaginations were seen on the outer membrane resembling microville and probably represented the remains of cell-to-cell interdigitations between hepatocytes and sinusoidal lining cells from the space of Disse. The cytoplasmic injury seen after Hanks' perfusion was reversed after collagenase perfusion. After mechanical dispersion, isolated mouse hepatocytes were spherical in shape and existed as individual cells; many (80 to 85%) were binucleated under hase contrast light microscopy. By electron microscopy, cells appeared morphologically similar in cytoplasmic constitution to that seen in intact nonaltered liver cells.

http://mouselivercells.com/Images/1981_Mouse%20Liver%20Cell%20Culture%20I_Hepatocyte%20Isolation_In%20Vitro.pdf

Mouse liver cell culture. I. Hepatocyte isolation.

The combination of interferon (IFN) and conventional chemotherapeutic agents offers a promising therapeutic approach for the treatment of cancer. However, there is as yet no consensus on optimal strategies for combining this family of compounds with other cancer therapies. While in vitro studies have demonstrated both direct cytotoxic and cytokinetic effects for IFN, a more interesting role derives from its ability to synergistically potentiate the activity of a wide variety of cytotoxic agents against multiple human and rodent tumors, both in vitro and in animal models. The interaction between IFN and cytotoxic agents in vitro is complex and depends not only on the choice of cytotoxic agent but also on the concentrations, ratios, duration, and sequence of exposure to the two drugs. Preliminary data suggest that some combinations are not merely additive but rather that IFN may biochemically modulate the cellular uptake or metabolism of the cytotoxic agent resulting in synergistic antineoplastic activity. In vivo interactions between IFN and cytotoxic agents involve an additional layer of complexity because of the potential effects of the biological agent on the host immune system and drug-metabolizing enzymes. Furthermore, IFN may have a protective effect on normal host tissues which theoretically could allow for the delivery of higher doses of cytotoxic agents. The results of early clinical trials using combinations of IFN with chemotherapeutic agents have generally been disappointing. This may be due to the inability of preclinical models to accurately predict the clinical situation or alternatively from a failure to incorporate information on dose, scheduling, and sequence of drug administration into clinical trials. Preliminary clinical studies with IFN-alpha and the fluorinated pyrimidine, 5-fluorouracil, in patients with advanced colorectal carcinoma suggest that IFN may enhance the effects of the antimetabolite. Confirmatory trials are in progress. Further trials designed to exploit the preclinical experience with combinations of IFN and cytotoxic agents are warranted.

https://cancerres.aacrjournals.org/content/canres/50/12/3473.full.pdf

Antineoplastic activity of the combination of interferon and cytotoxic agents against experimental and human malignancies: a review.

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 07 meeting in Washington, DC. The presentations discussed the phenomenology, clinical consequences, and underlying mechanisms of cytochrome P450 and drug transporter regulation by inflammatory and infectious stimuli. Although considerable insights into the links between inflammatory mediators and altered hepatic drug clearance pathways have been gained from previous studies with acute inflammatory stimuli, this symposium highlighted recent advances in understanding how these processes operate in other organs and chronic inflammatory states relevant to human diseases. The development of mouse models of live bacterial infection provides excellent opportunities to explore the impact of infection on drug metabolism beyond the well characterized effects of bacterial endotoxin. Altered levels of cytochromes P450 and especially drug transporters due to inflammation in brain, intestine, and placenta have significant implications for the use of many drugs in diverse clinical settings. The consequences of inflammatory cytokine production by tumors for drug safety and efficacy in cancer patients were outlined. Repression of drug clearance pathways by tumor-derived cytokines may result in extreme toxicity to chemotherapy, compromising treatment of many cancers. It is fitting that, in honoring the career contributions and achievements of Dr. Kenneth W. Renton, this symposium reinforced the clinical relevance of this field.

Regulation of Drug Metabolizing Enzymes and Transporters in Infection, Inflammation, and Cancer

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents.

Duration and intensity of drug action are greatly influenced by the rates of which drugs are biotransformed by the cytochrome P-450-linked monooxygenase systems of the hepatic endoplasmic reticulum. Several interferon-inducing agents (poly rI.rC, tilorone, vaccines, viruses, endotoxin) are shown to markedly depress hepatic P-450 systems when administered to rodents. The interferon (IF) inducers that depress hepatic drug metabolism also modulate certain immune responses; it is therefore not known whether the depression of P-450 is due to IF per se or to the action of IF-inducing agents on one or more components of the immune system. The loss of cytochrome P-450 elicited by IF-inducing agents is accompanied by a perturbation of heme metabolism associated with the dissociation of heme from cytochrome P-450. The agents also cause losses of hepatic catalase and tryptophan 2,3-dioxygenase. These studies predict that viral infections, vaccinations, and treatment with IF-inducing agents will be shown to seriously impair the metabolism of drugs in humans.

Effects of interferon‐inducing agents on hepatic cytochrome p‐450 drug metabolizing systems*

Previous studies from our laboratory have shown that a variety of interferon-inducing agents depress cytochrome P-450-dependent monooxygenase systems when administered to rats With the expectation that the use of cultured hepatocytes would provide a more accessible means of studying the mechanism by which interferon-inducing agents depress these enzyme systems, measurements were made of the effects of the interferon inducer, poly rI·rC and a crude preparation of mouse interferon on the cytochrome P-450 content and aminopyrine N-demethylase and benzo[a]pyrene hydroxylase activities of primary, nonreplicating mouse hepatocytes maintained on floating collagen membranes During the first 24 hr of culture, hepatocytes lost about 80% of their cytochrome P-450, 97% of their aminopyrine N-demethylase activity, and 90% of their benzo[a]pyrene hydroxylase activity The specific activity of cytochrome P-450 (nanomoles of [14C]formaldehyde formed from the N-demethylation of aminopyrine per nanomole of P-450 per minute) was lowered from 46 to as little as [unknown] this value Exposure of the cultures to poly rI·rC (5 µg/ml of culture) during the second 24 hr of culture caused a 40% increase in the cytochrome P-450 content of the hepatocytes The specific activity of cytochrome P-450 de novo relative to the N-demethylation of aminopyrine was restored to that of the cytochrome P-450 of freshly isolated hepatocytes or the cytochrome P-450 of microsomes isolated from liver homogenates The mouse interferon preparation (1000 units/ml of culture medium) was considerably less potent as an inducer of cytochrome P-450 in cultured hepatocytes, but the specific activity of the cytochrome P-450 de novo induced by mouse interferon was as high as that of the cytochrome P-450 induced by poly rI·rC Relative to benzo[a]pyrene hydroxylase activity, the specific activity of the cytochrome P-450 that survived the first 24 hr of culture was about the same as that for the cytochrome P-450 of microsomes isolated from liver homogenates Both poly rI·rC and mouse interferon preparation induced the hydroxylase activity in 24-hr-old cultures of hepatocytes Poly rI·rC did not induce the hydroxylase activity in cultured Reuber hepatoma cells Poly rI·rC was shown to induce interferon activity in cultured hepatocytes Methods are described for the determination of aminopyrine N-demethylase and benzo[a]pyrene hydroxylase activities and cytochrome P-450, cytochrome b5, and DNA contents of cultured hepatocytes from one mouse

Effects of polyribonoinosinic acid polyribocytidylic acid and a mouse interferon preparation on cytochrome P-450-dependent monooxygenase systems in cultures of primary mouse hepatocytes.

The cytochrome P -450 and heme contents of microsomes are lowered markedly in livers of rats treated with the interferon inducing agents, tilorone and poly(rI·rC) (polyriboinosinic acid·polyribocytidylic acid). The turnover of cytochrome P -450 heme was studied to determine whether the loss of cytochrome P -450 and heme was due to a decreased synthesis or to an increased degradation of the hemoprotein. Two populations of cytochrome P -450 exist in hepatic microsomes, one with a relatively short and one with a long half-life. The half-lives of cytochrome P -450 heme in control animals were determined to be about 8 and 40 h. After the administration of tilorone or poly(rI·rC), the half-life of the short-lived cytochrome P -450 was lowered to about 5 h but that of the long-lived cytochrome P -450 was not changed. These interferon inducing agents produced no appreciable decrease in the incorporation of the radioactivity of administered ALA-3,5-3H or glycine-2-14C into the heme of cytochrome P -420 derived from cytochrome P -450. However, a rise in specific activity was observed 10 and 24 h after the administration of poly(rI·rC) and tilorone, respectively. This increase in specific activity is interpreted to mean that interferon inducing agents increase the rate of breakdown of preformed cytochrome P -450 heme and that some of this is replaced by newly synthesized labeled heme. It is concluded from these results that poly(rI·rC) and tilorone lower the concentration of hepatic hemoproteins by increasing their degradation rather than by depressing their synthesis.

Effect of Interferon Inducing Agents (Polyriboinosinic Acid· Polyribocytidylic Acid and Tilorone) on the Heme Turnover of Hepatic Cytochrome P-450

Suppression of the inductive effects of phenobarbital and 3-methylcholanthrene on ascorbic acid synthesis and hepatic cytochrome P-450-linked monooxygenase systems by the interferon inducers, poly rI.rC and tilorone

Kenneth W. Renton

Papers

Depression of hepatic cytochrome P-450-dependent monooxygenase systems with administered interferon inducing agents.

Effects of interferon‐inducing agents on hepatic cytochrome p‐450 drug metabolizing systems*

Effects of polyribonoinosinic acid polyribocytidylic acid and a mouse interferon preparation on cytochrome P-450-dependent monooxygenase systems in cultures of primary mouse hepatocytes.

Effect of Interferon Inducing Agents (Polyriboinosinic Acid· Polyribocytidylic Acid and Tilorone) on the Heme Turnover of Hepatic Cytochrome P-450

Suppression of the inductive effects of phenobarbital and 3-methylcholanthrene on ascorbic acid synthesis and hepatic cytochrome P-450-linked monooxygenase systems by the interferon inducers, poly rI.rC and tilorone