Jeffrey R. Fry

Influence of cytochrome P-450 type on the pattern of conjugation of 7-hydroxycoumarin generated from 7-alkoxycoumarins

Pretreatment of rats with phenobarbitone increased hepatic microsomal 7-methoxy-and 7-ethoxy-coumarin O-dealkylase activities. Pretreatment with beta-naphthoflavone increased only the 7-ethoxycoumarin O-dealkylase activity. The addition of metyrapone in vitro inhibited the O-dealkylations to different extents. Similar results were obtained with diphenyloxazole and ethanol. These results are taken to indicate that different forms of cytochrome P-450 are involved in the O-dealkylation of these two substrates. The pattern of metabolism (Phase I and Phase II) of each alkoxycoumarin in rat isolated hepatocytes was very similar. The sulphate conjugate was the major metabolite produced, the amount of which approached a plateau as the rate of O-dealkylation increased. It is concluded that the type of cytochrome P-450 involved in the initial Phase I metabolism does not influence the subsequent pattern of conjugation.

Detection of reactive metabolites in vitro

The ability of 27 compounds to mediate depletion of glutathione (GSH) in a fortified liver microsome incubation via production of reactive metabolites generated by microsomal mono-oxygenase (MMO) metabolism has been studied. The majority of compounds tested of this type were positive in this assay, with the exception of iproniazid and naphthalene. Thioacetamide, the reactive metabolite of which binds via lysine residues, was negative. Acrolein and hexachlorobutadiene produced depletion in the absence of prior activation, as did menadione but for which enzyme induction increased the magnitude of depletion. Allyl alcohol produced MMO-mediated depletion of GSH. Some depletion of GSH occurred in the presence of common substrates of the MMO system. It is suggested that this depletion assay may be a valid test for detection of reactive metabolites generated by MMO metabolism.

Metabolism of coumarin by rat, gerbil and human liver microsomes

1. o-Hydroxyphenylacetaldehyde was the major metabolite of coumarin (1 mM) in rat, gerbil and human liver microsomes. 2. Treatment of rats with phenobarbitone (PB) or beta-naphthoflavone increased the o-hydroxyphenylacetaldehyde formed. 3-Hydroxycoumarin was the other main metabolite produced by rat liver microsomes. 3. Liver microsomal metabolism of coumarin in gerbil was extensive with 3-, 5-, 6-, 7- and 8-hydroxycoumarins, and 3,7- and 6,7-dihydroxycoumarins produced, in addition to o-hydroxyphenylacetaldehyde. The profile of the hydroxy metabolites was altered by in vivo treatment of gerbils with cytochrome P-450 inducers, but there was no increase of coumarin metabolism. 4. Coumarin was metabolized by human liver microsomes to o-hydroxyphenylacetaldehyde, 7-hydroxycoumarin, 3-hydroxycoumarin, and trace amounts of 5-, 6- and 8-hydroxycoumarins. 5. At low substrate concentrations (0-10 microM) hepatic microsomal metabolism of coumarin in gerbil resembled that in man, with 7-hydroxycoumarin being a major metabolite. However, the production of o-hydroxyphenylacetaldehyde was greater in gerbil than human liver microsomes. 6. At higher substrate concentrations (1 mM) metabolism of coumarin by liver microsomes from PB-treated gerbils most closely resembled that by human liver microsomes. 7. The gerbil would appear to be a more appropriate animal model than rat for studies to assess the toxicological hazard of coumarin for man.

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.

Toxicity and association of polycyanoacrylate nanoparticles with hepatocytes.

The degradation of polybutylcyanoacrylate (PBC) and polyhexylcyanoacrylate (PHC) nanoparticles, together with their association with and toxicity towards isolated hepatocytes, were determined. Nanoparticles were not taken up by rat hepatocytes at a significant level. The LD50S of PBC and PHC nanoparticles towards hepatocytes were 0.4 mg/2 x 10(6) cells and greater than 1 mg/2 x 10(6) cells respectively. This hepatocyte toxicity cannot be attributed solely to the formaldehyde formed during degradation.