Alan J. Paine

Apparent lack of correlation between the loss of cytochrome P-450 in hepatic parenchymal cell culture and the stimulation of haem oxygenase activity

Abstract The hypothesis that the stimulation of haem oxygenase activity in cultured adult rat liver parenchymal cells is intimately associated with the accelerated breakdown of the haemoprotein cytochrome P-450 was examined. Even though the time course of the loss of cytochrome P-450 and the stimulation of haem oxygenase activity were found to be compatible with this hypothesis, further work however showed that high levels of cytochrome P-450 could be maintained in liver cell culture in the face of high haem oxygenase activities.

Apparent maintenance of cytochrome P 450 by nicotinamide in primary cultures of rat hepatocytes

Abstract The sole addition of a high, unphysiological, concentration of nicotinamide (25 mM) to a cell culture medium was found to maintain the cytochrome P 450 concentration of rat hepatocytes cultured for 24 hours at 71% of the level found in intact liver, whilst hepatocytes cultured without nicotinamide contained only 20% of their initial cytochrome P 450. Furthermore the P 450 concentration of hepatocytes cultured for 24 hours in the presence of 25 mM nicotinamide could be increased to the same level as found in intact rat liver by the inclusion of 1 mM nicotinamide into the medium used for cell isolation. Although the mechanism of action of nicotinamide is unknown this simple system for the maintenance of cytochrome P 450 in hepatocyte culture could provide the opportunity to study, under defined conditions in vitro , the factors that regulate cytochrome P 450 and hence determine hepatotoxicity and hepatocarcinogenesis.

A comparison of the accumulation of ricin by hepatic parenchymal and non-parenchymal cells and its inhibition of protein synthesis

Rat liver non-parenchymal cells in vivo were found to accumulate 125I-labelled ricin to a much greater extent than parenchymal cells. Similarly, in monolayer cell cultures, the rate of ricin uptake by non-parenchymal Kupffer cells was several times that by parenchymal cells. Evidence is provided also to suggest that ricin is primarily recognized by Kupffer cells via terminal mannose residues in the toxin, whereas ricin uptake by parenchymal cells was consistent with a role of the previously postulated galactosyl-containing cell receptors. Protein synthesis in Kupffer cells in vitro, although observed to occur at a lower rate than in parenchymal cells, was 100--1000-times more sensitive to inhibition by ricin. The selective damage known to be caused to liver sinusoids by ricin, therefore, may reflect both the relative efficiency with which the toxin is taken up by these cells and the extreme sensitivity of protein synthesis in the cells to inhibition by ricin.

The relationship between the ability of pyridine and substituted pyridines to maintain cytochrome P-450 and inhibit protein synthesis in rat hepatocyte cultures.

Abstract In order to gain an insight into the mechanism of maintenance of cytochrome P-450 by nicotinamide (pyridine-3-carboxylic acid amide) in liver cell culture, the ability of pyridine and substituted pyridines to maintain cytochrome P-450 were compared. The structure-activity relationship suggests that the feature of nicotinamide important for the maintenance of cytochrome P-450 is a primary amide group attached to a pyridine ring. Pyridine itself, but not secondary or tertiary amides of pyridine, is also able to maintain cytochrome P-450. To examine the hypothesis that the ability of compounds to maintain cytochrome P-450 in liver cell culture may be related to their ability to inhibit protein synthesis, the effect of pyridine analogues and cycloheximide were compared. The results suggest that the inhibition of protein synthesis is not a mechanism underlying the maintenance of cytochrome P-450 in hepatocyte culture.

Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake

The relative toxicities of particulate beryllium phosphate, soluble beryllium sulphate and a beryllium sulphosalicylate complex to a rat liver parencymal derived cell line have been examined in culture. Due to the propensity of beryllium salts to form beryllium phosphate in solution the incubation medium used was free of inorganic phosphate. Cell death measured by the loss of cellular lactate dehydrogenase into the medium can be produced within 76 h from beryllium phosphate and beryllium sulphosalicylate or 48 h from beryllium sulphate provided the cells have, irrespective of the form of added beryllium, taken up a minimum of 2--5 nmol Be/10(6) cells. Whilst beryllium phosphate was readily taken up as a particle, beryllium complexed with excess sulphosalicylate was not so markedly accumulated by the cells except possibly by formation of small amounts of beryllium phosphate in the medium as a result of inorganic phosphate lost from the cells. The extent of beryllium uptake from beryllium sulphate quantitatively most resembled that observed for beryllium phosphate but was largely independent of beryllium phosphate formation in the medium and not accompanied by the uptake of the SO42- anion. However, the accumulation of beryllium derived from beryllium sulphate did appear to be associated with the production of a sedimentable from believed most probably to be colloidal beryllium hydroxide. The uptake of all forms of beryllium was temperature sensitive and metabolic inhibitor studies and treatment of the cells with trypsin or neuraminidase supported the view that the distinct behaviour of beryllium derived from beryllium sulphate may be related to the enhanced toxicity of this form both under the conditions used and when administered to experimental animals.

The induction of benzo[a]pyrene-3-mono-oxygenase by singlet oxygen in liver cell culture is mediated by oxidation products of histidine

The photochemical generation of excited states of oxygen by the mild illumination of culture medium containing 15 microM riboflavin results in a typical induction of benzo[a]pyrene-3-mono-oxygenase in cell lines derived from liver. However, the induction of the mono-oxygenase is not due to an excited state of oxygen directly activating the inducing mechanism inside the cell but is due to the oxidation of a component of the culture medium forming a stable inducer. The present work unequivocably shows that the component oxidised is histidine. The mild illumination of culture medium containing riboflavin therefore converts a physiological component of the medium which is not normally an inducer of the mono-oxygenase into a compound which is as effective an inducer as the classical inducer. The finding that singlet oxygen will oxidise a cell constituent into a powerful inducer is compatible with the hypothesis that excited states of oxygen and their oxidation products may play a central role in the induction of cytochrome P-450 and associated enzyme activities by many chemically unrelated inducers.

Ligands maintain cytochrome P-450 in liver cell culture by affecting its synthesis and degradation.

Abstract Rat hepatocytes cultured for 24 h lose 68% of their cytochrome P-450. It is shown that this loss is due to the failure of cultured hepatocytes to synthesize cytochrome P-450 as well as enhanced degradation. Compounds that form ligands with cytochrome P-450, eg metyrapone, prevent the loss of cytochrome P-450. Ligands are generally considered to protect proteins from degradation but the present work suggests that the effect of metyrapone on cytochrome P-450 synthesis is of equal importance to its effect on degradation in preventing the loss of cytochrome P-450 in hepatocyte culture.

Attachment of rat hepatocytes to plastic substrata in the absence of serum requires protein synthesis.

Abstract Rat hepatocytes attach within 1 hour to plastic petri dishes if foetal calf serum is added to the culture medium. In the absence of serum rat liver cells attach only after a lag phase of 4 hours. However hepatocyte monolayers are formed under either culture condition demonstrating that hepatocyte culture does not have an absolute requirement for foetal calf serum. Cycloheximide is without effect on cell attachment in serum containing medium which is compatible with the concept that attachment is mediated by a “cold insoluble globulin” present in foetal calf serum. In contrast cycloheximide markedly inhibits the attachment of hepatocytes cultured without serum suggesting that rat liver cells are capable of making their own “attachment” factor. This factor appears to be released into the medium and has a similar sensitivity to heat and acid treatment as the “attachment” protein present in foetal calf serum.

Comparison of the photochemical and enzymic generation of excited states of oxygen on the induction of benzo(α)pyrene mono-oxygenase in liver cell cultures

The photochemical generation of excited states of oxygen such as the superoxide ion(O-2) and singlet oxygen (1o2) by the mild illumination of culture medium containing riboflavin induces benzo(alpha)pyrene mono-oxygenase in 3 different cell lines derived from rat liver. Similar rates of O-2 generation can be produced by the action of xanthine oxidase on xanthine yet this system does not induce the mono-oxygenase. This result confirms that the mono-oxygenase induction is not mediated by O-2 is not mediated by O-2 and that 1O2 is the most likely candidate for stimulating the mono-oxygenase activity.