Alison H. Hammond

Liver Tissue Engineering: A Role for Co-culture Systems in Modifying Hepatocyte Function and Viability

A major limitation in the construction of a functional engineered liver is the short-term survival and rapid de-differentiation of hepatocytes in culture. Heterotypic cell-cell interactions may have a role to play in modulating long-term hepatocyte behavior in engineered tissues. We describe the potential of 3T3 fibroblast cells in a co-culture system to modulate function and viability of primary isolated rat hepatocytes. Over an 18-day period after isolation, hepatocytes in pure culture rapidly declined in viability, displayed sparse bile canaliculi, and lost two function markers, the secretion of albumin and ethoxyresorufin O-dealkylase (EROD) activity. In comparison, the hepatocytes within the co-cultures maintained viability, possessed well-formed canalicular systems, and displayed both functional markers. Fixed 3T3 cells or 3T3 cell conditioned medium did not substitute for the viable 3T3 cell co-culture system in preserving hepatocyte viability and functionality.

Involvement of cytochrome P4502E1 in the toxicity of dichloropropanol to rat hepatocyte cultures

Hepatocytes were isolated and cultured from untreated rats and rats treated with isoniazid to induce cytochrome P4502E1. Isoniazid selectively increased p-nitrophenol hydroxylase activity in 2-h cultures, and increased the toxicity of both 1,3- and 2,3-dichloropropanol. Isoniazid also increased the rate and extent of glutathione depletion by the dichloropropanols. There was no effect of isoniazid on the toxicity of 1,3-dichloroacetone, precocene II or allyl alcohol. In addition, diethyldithiocarbamate selectively inhibited p-nitrophenol hydroxylase in 2-h cultures from untreated and isoniazid-treated rats, as well as abolishing toxicity of the dichloropropanols. In 24-h cultures from isoniazid-treated rats diethyldithiocarbamate inhibited high affinity MCOD activity by 55% and there was also a small but significant inhibition of precocene II toxicity. These results indicate that isoniazid-inducible P4502E1 can mediate the toxicity of dichloropropanol.

EFFECT OF CYANAMIDE ON TOXICITY AND GLUTATHIONE DEPLETION IN RAT HEPATOCYTE CULTURES : DIFFERENCES BETWEEN TWO DICHLOROPROPANOL ISOMERS

The effect of aldehyde dehydrogenase inhibition by cyanamide pre-treatment in vitro on dichloropropanol-dependent toxicity and glutathione depletion was investigated in 24 h rat hepatocyte cultures. Cyanamide pre-treatment had no effect on nitrophenol hydroxylase, 7-methoxy-, 7-ethoxy- or 7-benzyloxyresorufin O-dealkylase activities in 24 h cultures from untreated rats, and had no effect on intracellular glutathione content in cultures from untreated rats, or in cultures from isoniazid-treated rats in which cytochrome P4502E1 (CYP2E1) is increased. In cultures from untreated animals the primary alcohol, 2,3-dichloropropanol, was not toxic and did not significantly deplete glutathione. Cyanamide pre-treatment however, potentiated both toxicity and glutathione depletion. Induction of CYP2E1 also potentiated the toxicity of 2,3-dichloropropanol, and in these cultures cyanamide pre-treatment significantly increased both toxicity and glutathione depletion. Cyanamide did not alter the toxicity or glutathione depletion due to the secondary alcohol, 1,3-dichloropropanol, irrespective of CYP2E1 induction. These results indicate that the primary alcohol isomer is metabolised to an aldehyde intermediate which depletes glutathione. Under basal conditions this metabolite appears to be effectively detoxified, but increased CYP2E1 activity and/or decreased aldehyde dehydrogenase activity promotes accumulation of metabolite, and therefore increases glutathione depletion and toxicity.

Toxicity of dichloropropanols in rat hepatocyte cultues.

Exposure of humans to dichloropropanols has been shown to result in fulminant hepatic necrosis. These compounds have also been shown to be hepatotoxic in rats. In this study, 1,3-dichloropropanol, but not 2,3-dichloropropanol, was shown to be toxic to 24 h cultures of rat hepatocytes. The toxicity was inhibited by pre-treatment of cultures with a cytochrome P450 inhibitor and enhanced by prior depletion of cellular glutathione. In addition, at equimolar concentrations both isomers were shown to deplete glutathione, although the extent of depletion was greater with the 1,3-isomer. 1,3-Dichloropropanol also depleted ATP and reduced the mitochondrial membrane potential. The effects on ATP, glutathione and membrane potential could be inhibited by the cytochrome P450 inhibitor. It is concluded that the toxicity of 1,3-dichloropropanol is mediated by cytochrome P450 and involves depletion of glutathione and loss of mitochondrial function.

Effect of serum-free medium on cytochrome P450-dependent metabolism and toxicity in rat cultured hepatocytes.

Cytochrome P450 (P450)-dependent activities in homogenates of rat hepatocytes cultured for 96 hr in serum-free and serum-containing medium were compared. Benzphetamine and erythromycin N-demethylases, 7-methoxy- and propoxy-coumarin O-dealkylases, p-nitrophenol hydroxylase and 7-ethoxy- and pentoxy-resorufin O-dealkylases were all maintained at higher levels in hepatocytes cultured in serum-free medium, although there was some selectivity with respect to the extent of the maintenance relative to the activities in fresh cells. The toxicities of coumarin, precocene I and precocene II to 24 hr hepatocyte cultures, determined as decreased survival, were also shown to be increased in serum-free medium. However, the magnitude of the difference between media with respect to the toxicity of precocene II was decreased in hepatocytes cultured for 72 hr. The observed increase in toxicity is consistent with the improved maintenance of P450 in hepatocytes cultured in serum-free medium, although there is still a selective decline in P450 activities and toxicity with increased time in culture. The activity of alcohol dehydrogenase and the toxicity of allyl alcohol were similar in hepatocytes cultured in serum-free and serum-containing medium for 96 hr. The absence of serum did not affect the non-protein sulphydryl content of the cultures.

Correlation of acute lethal potency with in vitro cytotoxicity

The in vitro cytotoxicity (measured as ID(50) value) of 27 compounds believed to act by interference with cell basal functions/structures has been determined and compared with their acute lethal toxicity (measured as oral and ip LD(50) values) using a previously described approach to the choice of the most appropriate LD(50) value from the Registry of Toxic Effects of Chemical Substances database. A weak, but significant, positive correlation of log oral LD(50) value with log ID(50) value was obtained (r = 0.49; 0.02 < P < 0.05) for 21 compounds for which oral and ip LD(50) values were obtainable. A better correlation between LD(50) and ID(50) was obtained when the log ip LD(50) value was used (r = 0.68; P < 0.001). Three compounds, for which metabolism is a major determinant of toxicity in vivo, yielded anomalous results in the ip LD(50)/ID(50) comparison. A further improvement in the correlation of log ip LD(50) with log ID(50) was obtained when data from these compounds were excluded (r = 0.82; P < 0.001). Close correlations of log ip LD(50)/log ID(50) were obtained with groups of six anti-metabolites and six alkylating agents (r = 0.94 and 0.96, respectively; P < 0.001 in each case). The locations of the regression lines for these two groups were significantly different (P < 0.01). It is concluded that the in vitro cytotoxicity of compounds that exert their toxicity by interference with cell basal functions/structures is correlated with their intrinsic lethal potency, but that information on absorption, metabolism and disposition are required before the cytotoxicity data can be used to assess apparent potency. The data provide evidence that the precise relation of LD(50)/ID(50) values is determined by the mode of toxicity.

Toxicity of coumarin and various methyl derivatives in cultures of rat hepatocytes and V79 cells.

The toxicity of coumarin and various simple methyl derivatives in rat hepatocyte and V79 cell cultures was studied to investigate further the mechanism of coumarin hepatotoxicity. Coumarin was six times more toxic in hepatocyte cultures from phenobarbitone (PB)-treated rats than in those from untreated rats. At concentrations below 3 mm, coumarin did not affect the survival of V79 lung fibroblasts. SKF-525A inhibited coumarin-induced toxicity in hepatocytes cultured from PB-treated rats, whereas depletion of hepatocyte glutathione (GSH) levels with buthionine sulphoximine (BSO) significantly increased toxicity. Dihydrocoumarin (DHC) had little effect on the survival of cultured hepatocytes, indicating that the 3,4-double bond is an important determinant of coumarin toxicity. In general, the toxicity of coumarin in hepatocyte cultures was reduced by substitution with one or more methyl groups. 3-Methylcoumarin (MeC), however, was more toxic than coumarin itself in hepatocyte cultures from untreated rats. Except for 3,4-diMeC, the methyl derivatives were markedly more toxic in rat hepatocytes than in V79 cell cultures. The data obtained for coumarin and 4-MeC, and possibly 6-MeC and 7-MeC, are consistent with hepatocyte toxicity being due to the cytochrome P-450-dependent formation of one or more toxic metabolites that may be detoxified by reacting with GSH. This was less apparent for 3-MeC and 3,4-diMeC, although depletion of GSH levels significantly increased the hepatocyte toxicity of both compounds.

The in vivo induction of rat hepatic cytochrome P450-dependent enzyme activities and their maintenance in culture

Cytochrome P450-dependent enzyme activities were measured in hepatocytes from adult male rats, induced in vivo with phenobarbitone, beta-naphthoflavone, dexamethasone or isoniazid: the stability of the induced activities in culture was also determined. Each inducer produced a characteristically different pattern of enzyme activities with dexamethasone, isoniazid and beta-naphthoflavone selectively inducing erythromycin N-demethylase, p-nitrophenol hydroxylase and ethoxyresorufin O-dealkylase respectively. In general, the induced activities were maintained for 24 hr in culture. This indicates the feasibility of an in vivo induction-hepatocyte culture system for the study of metabolism-mediated toxicity.

Comparison of in vivo and in vitro rat hepatic toxicity of coumarin and methyl analogues, and application of quantitative morphometry to toxicity in vivo

The rat hepatic toxicity of coumarin and methyl analogues (3-,4-methyl coumarin and 3,4-dimethylcoumarin) has been determined in vivo and in vitro (freshly-isolated cells). Coumarin at a dose of approximately 1 mmol/kg produced clear histological evidence of centrilobular necrosis, while the methyl analogues at an equivalent dose were much less toxic. By use of a systematic random sampling protocol and quantitative morphometry it was determined that there was a lobar variation in the extent of hepatic damage but that this exhibited random inter-animal variation. The order of cytotoxicity in vitro was identical to that observed in vivo. In hepatocytes depleted of glutathione the toxicity of all four compounds was increased. This was particularly marked for the 3-methyl analogues, such that the order of toxicity was different to that observed in vivo and in hepatocytes not depleted of glutathione.

Maintenance of xenobiotic metabolism and toxicity in rat hepatocyte cultures after cell preservation at 4

Isolated rat hepatocytes were stored at 4 degrees in modified University of Wisconsin solution for 22-24 hr and then placed into culture. Satisfactory viabilities were obtained for all preparations. No loss of alcohol dehydrogenase or 7-ethoxy-3,4-dimethylcoumarin O-deethylase activity was observed in the cold stored relative to the control cultures. The protein and glutathione contents, allyl alcohol and precocene II (PII) toxicities were also similar, as was the PII-mediated glutathione depletion. For the parameters measured, cultures of cold stored hepatocytes were identical to normal cultures.