Roger J. Price

Inhibition of xenobiotic-induced genotoxicity in cultured precision-cut human and rat liver slices

In this study precision-cut liver slices have been used to evaluate the effects of the flavone tangeretin, the flavonoid glycoside naringin and the flavanone naringenin (the aglycone derived from naringin) on xenobiotic-induced genotoxicity. Liver slices were cultured for 24 h in medium containing [3H]thymidine and the test compounds and then processed for autoradiographic determination of unscheduled DNA synthesis (UDS). The cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) markedly induced UDS in cultured human liver slices and both 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1) induced UDS in cultured rat liver slices. Tangeretin (20 and 50 microM) was found to be a potent inhibitor of 5 and 50 microM PhIP-induced UDS in human liver slices, whereas 20 and 50 microM naringenin was ineffective and naringin only inhibited genotoxicity at a concentration of 1000 microM. In rat liver slices 50 microM tangeretin inhibited 10 and 50 microM 2-AAF-induced UDS, whereas 50 microM naringenin and 100 and 1000 microM naringin were ineffective. None of the three flavonoids examined inhibited 5 microM AFB1-induced UDS in rat liver slices. The inhibition of PhIP- and 2-AAF-induced UDS by tangeretin is probably attributable to the inhibition of the human and rat cytochrome P-450 isoforms which are responsible for the bioactivation of these two genotoxins. Although flavonoids can modulate xenobiotic-induced genotoxicity in human and rat liver slices, any protective effect is dependent on the particular combination of genotoxin and flavonoid examined. These results demonstrate that cultured precision-cut liver slices may be utilised as an in vitro model system to examine the modulation of xenobiotic-induced genotoxicity by flavonoids and other dietary components.

Modification of the carbohydrate in ricin with metaperiodate and cyanoborohydride mixtures: effect on binding, uptake and toxicity to parenchymal and non-parenchymal cells of rat liver

The carbohydrate in the toxic glycoprotein ricin was chemically modified by simultaneous treatment with sodium metaperiodate and sodium cyanoborohydride. This treatment causes oxidative cleavage of the sugar residues and reduction of the aldehyde groups which are formed to primary alcohols. The modification markedly decreased the rapid removal of ricin from the blood by hepatic non-parenchymal cells with only a relatively small increase in accumulation of the toxin by parenchymal cells. Binding, uptake and toxicity of the modified ricin in primary monolayer cultures of hepatic non-parenchymal cells were all decreased to a much greater extent than in parenchymal cells. The results indicate that native ricin binds to non-parenchymal cells by a dual recognition process which involves both interaction of cell receptors with the mannose-containing oligosaccharides of the toxin and binding of ricin to galactose-containing glycoproteins and glycolipids on the cells. However, uptake and toxicity of native ricin in non-parenchymal cells appears to result principally from entry of the toxin through the mannose recognition pathway. By contrast, uptake and toxicity of the expressed essentially through the galactose-recognition route.

Uptake of native and deglycosylated ricin A-chain immunotoxins by mouse liver parenchymal and non-parenchymal cells in vitro and in vivo

The therapeutic activity of ricin A-chain immunotoxins is undermined by their rapid clearance from the bloodstream of animals by the liver. This uptake has generally been attributed to recognition of the mannose-terminating oligosaccharides present on ricin A-chain by receptors present on the non-parenchymal (Kupffer and sinusoidal) cells of the liver. However, we demonstrate here that, in the mouse, the liver uptake of a ricin A-chain immunotoxin occurs in both parenchymal and non-parenchymal cells in equal amounts. This is in contrast to the situation in the rat, where uptake of the immunotoxin is predominantly by the non-parenchymal cells. Recognition of sugar residues on the A-chain portion of the immunotoxin plays an important role in the liver uptake by both cell types in both species. However it is not the only mechanism since, firstly, an immunotoxin containing ricin A-chain which had been effectively deglycosylated with metaperiodate and cyanoborohydride was still trapped to a significant extent by hepatic non-parenchymal cells after it was injected into mice. Secondly, deglycosylation, while eliminating uptake of the free A-chain by parenchymal and non-parenchymal cells in vitro, only reduced the uptake of an immunotoxin by either cell type by about half. Thirdly, the addition of excess D-mannose or L-fucose inhibited the uptake of free A-chain by mouse liver cell cultures by more than 80% but only inhibited the uptake of the native A-chain immunotoxin by about half and had little effect on the uptake of the deglycosylated ricin A-chain immunotoxin. Recognition of the antibody portion of the immunotoxin by liver cells seems improbable, since antibody alone or an antibody-bovine serum albumin conjugate were not taken up in appreciable amounts by the cultures. Possibly attachment of the A-chain to the antibody exposes sites on the A-chain that are recognised by liver cells in vitro and in vivo.

Lack of effect of piperonyl butoxide on unscheduled DNA synthesis in precision-cut human liver slices

In this study the effect of piperonyl butoxide (PBO) on unscheduled DNA synthesis in precision-cut human liver slices has been examined. Liver slices prepared from tissue samples from five human donors were cultured in medium containing [3H]thymidine and 0-2.5 mM PBO using a dynamic organ culture system. After 24 h the liver slices were processed for autoradiographic examination of UDS. As positive controls, liver slices were also cultured with three known genotoxic agents, namely 2-acetylaminofluorene (2-AAF), aflatoxin B1 (AFB1) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with > 5 and > 10 net grains. Compared to control liver slice cultures PBO had no effect on UDS. In contrast, treatment with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 mM AFB1 and 0.005 and 0.05 mM PhIP produced significant increases in net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 mM PhIP. Treatment with 2-AAF, AFB1 and PhIP also produced increases in the number of centrilobular hepatocyte nuclei with > 5 and > 10 net grains. At the concentrations examined neither PBO, 2-AAF nor PhIP had any significant effect on replicative DNA synthesis in 24 h cultured human liver slices. In cultured liver slices treated with 0.02, but not 0.002, mM AFB1 a significant reduction in the rate of replicative DNA synthesis was observed. These results demonstrate that PBO does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice preparations used in this study. In conclusion, these results provide further evidence that PBO is a non-genotoxic agent which does not damage DNA in human liver.

Comparison of the toxicity of allyl alcohol, coumarin and menadione in precision-cut rat, guinea-pig, Cynomolgus monkey and human liver slices

Abstract The toxicity of allyl alcohol, coumarin and menadione has been studied in precision-cut liver slice cultures. Liver slices were prepared from male Sprague- Dawley rats, male Dunkin-Hartley guinea-pigs and from samples of Cynomolgus monkey and human liver using a Krumdieck tissue slicer. The liver slices were cultured with the test compounds for 24h in a dynamic organ culture system. Toxicity was assessed by measurement of protein synthesis, potassium content and the MTT assay. At the concentrations examined, menadione produced marked toxicity in liver slices from all four species, whereas rat liver slices were less susceptible to allyl alcohol toxicity. Coumarin produced concentration-dependent toxic effects in rat and guinea-pig liver slices, whereas Cynomolgus monkey and human liver slices were relatively resistant, especially at low coumarin concentrations. At some concentrations of the test compounds examined, the MTT assay appeared to be a less sensitive indicator of toxicity than either protein synthesis or potassium content. These results demonstrate the usefulness of precision-cut liver slices for assessing species differences in xenobiotic-induced toxicity.

The uptake and subsequent loss of beryllium by rat liver parenchymal and non-parenchymal cells after the intravenous administration of particulate and soluble forms

Abstract A cell isolation technique has been used to study the uptake and subsequent loss of beryllium (Be) by rat liver after intravenous administration of non-lethal doses of either particulate beryllium phosphate or the more hepatotoxic soluble BeSO4. It has been shown that beryllium phosphate is removed from the blood predominantly by the non-parenchymal (sinusoidal) cells of the liver and to a lesser extent more slowly by the parenchymal cells. After 24 h when the parenchymal cells have reached maximal Be content there has been a 50% loss of Be from the non-parenchymal cells and a similar loss from whole liver which is reflected in an increased level of Be in the blood. The Be count of non-parenchymal cells subsequently decreases much more slowly in a manner similar to that of the parenchymal cells, both being only halved during the following week. Within 24–48 h some redistribution of Be to the spleen occurs and it is suggested that this in part may be the result of Kupffer cell death. In splenectomized animals a high proportion of this redistributed Be appears to be retaken up by the liver mainly by the parenchymal cell population. After administration of BeSO4, which is known to form beryllium phosphate in plasma, a greater proportion of the Be is taken up slowly by the parenchymal cells and no redistribution of Be to the spleen is observed. It is suggested that this behaviour is related primarily to the smaller size and nature of the beryllium phosphate particles formed in plasma under these conditions. The rate of loss of Be from both the parenchymal and non-parenchymal cells is similar to that measured in beryllium phosphate treated animals. It has been estimated that liver cell death is produced when the cell content exceeds 2–3 nmol Be/106 cells although parenchymal cells appear to be more sensitive to Be derived from BeSO4 than preformed beryllium phosphate.

Toxicity of 3-methylindole, 1-nitronaphthalene and paraquat in precision-cut rat lung slices.

The toxicity of 3-methylindole, 1-nitronaphthalene and paraquat has been studied in precision-cut rat lung slice cultures. Lung slices were prepared from male Sprague-Dawley rats using an agarose gel instilling technique with a Krumdieck tissue slicer and cultured for 24 h in a dynamic organ culture system. Treatment of rat lung slices with 3-methylindole, 1-nitronaphthalene or paraquat produced concentration dependent decreases in lung slice protein synthesis and potassium content. EC50 values (concentration to produce a 50% inhibition) for protein synthesis were 0.024, 0.27 and 0.57 mM for paraquat, 1-nitronaphthalene and 3-methylindole, respectively. These results demonstrate that precision-cut lung slices are a useful in vitro model system for studying the pulmonary toxicity of xenobiotics. Lung slices offer the potential as a rapid in vitro screen for identifying pulmonary toxicants and to evaluate species differences in response.

Mitogenic effects of beryllium and zirconium salts on mouse splenocytes in vitro

The beryllium (Be) and zirconium (Zr) salts, BeSO4 and Zr(SO4)2, each exerted a concentration-dependent stimulation of mouse spleen cell proliferation as measured by an increase in [3H]thymidine incorporation into lymphocyte DNA, although the maximal response induced by Zr(SO4)4 (4-5 fold at 100-200 microM) was greater than that by BeSO4 (2-3 fold at 1-5 microM). Preincubation of splenocytes with low concentrations of BeSO4 (less than 1 microM) or a broad range of Zr(SO4)2 concentrations (2-100 microM) was also found to assist subsequent lectin (concanavalin A; ConA)-mediated lymphocyte proliferation. The results indicate that at defined concentrations Be and Zr salts can both act as lymphocyte mitogens and augment the functional responsiveness of immune cells, which may help explain the characteristic induction of delayed hypersensitivity and production of immunological granulomas by these metals in vivo.

Mannose receptor dependent uptake of a ricin a chain - antibody conjugate by rat liver non-parenchymal cells

Mannose receptor mediated uptake by the reticuloendothelial system has been suggested as an explanation for the rapid removal of ricin A chain antibody conjugates from the circulation after their administration. We have measured, in the rat, hepatic uptake of a ricin A chain antibody conjugate in vivo and its susceptibility to inhibition by a mannosylated protein and have measured uptake of the conjugate in vitro by rat parenchymal and non-parenchymal liver cells. The results indicate that rapid hepatic uptake of conjugate does occur in vivo; cultured non-parenchymal cells accumulate the conjugate to a much greater degree than cultured parenchymal cells and that mannose receptors appear to be involved in the process.

In vitro assays for induction of drug metabolism.

Hepatic microsomal cytochrome P450 (CYP) forms have a major role in the metabolism of drugs and other chemicals. Primary hepatocyte cultures from humans and experimental animals are a valuable in vitro system for studying the effects of chemicals on CYP forms. This chapter describes methods to evaluate CYP form induction in human and rat hepatocytes cultured in a 96-well plate format. The use of a 96-well plate format permits studies to be performed with relatively small numbers of hepatocytes and obviates the need to harvest cells and prepare subcellular fractions prior to the assay of enzyme activities. The induction of CYP1A and CYP3A forms in human and rat hepatocytes can be determined by measurement of 7-ethoxyresorufin O-deethylase and testosterone 6beta-hydroxylase activities, respectively, whereas 7-benzyloxy-4-trifluoromethylcoumarin (BFC) O-debenzylase can be employed to assess both CYP1A and CYP2B form induction in rat hepatocytes. An assay for determining the protein content of hepatocytes cultured in a 96-well plate format is also described.