Predicted Michaelis-Menten complexes of cocaine-butyrylcholinesterase. Engineering effective butyrylcholinesterase mutants for cocaine detoxication

Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (-)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser(198) for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe(329) and Trp(430). These interactions do not occur with (+)-cocaine. Because the rate of (-)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (-)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.     

Relationships Between the Catechol Substrate Binding Site and Amphetamine, Cocaine, and Mazindol Binding Sites in a Kinetic Model of the Striatal Transporter of Dopamine In Vitro

Abstract: Experiments were conducted to determine how (−)-cocaine and S (+)-amphetamine binding sites relate to each other and to the catechol substrate site on the striatal dopamine transporter (sDAT). In controls, m -tyramine and S (+)-amphetamine caused release of dopamine from intracellular stores at concentrations ≥12-fold those observed to inhibit inwardly directed sDAT activity for dopamine. In preparations from animals pretreated with reserpine, m -tyramine and S (+)-amphetamine caused release of preloaded dopamine at concentrations similar to those that inhibit inwardly directed sDAT activity. S (+)-Amphetamine and m -tyramine inhibited sDAT activity for dopamine by competing for a common binding site with dopamine and each other, suggesting that phenethylamines are substrate analogues at the plasmalemmal sDAT. (−)-Cocaine inhibited sDAT at a site separate from that for substrate analogues. This site is mutually interactive with the substrate site ( K _int = 583 n M ). Mazindol competitively inhibited sDAT at the substrate analogue binding site. The results with (−)-cocaine suggest that the (−)-cocaine binding site on sDAT is distinct from that of hydroxyphenethylamine substrates, reinforcing the notion that an antagonist for (−)-cocaine binding may be developed to block (−)-cocaine binding with minimal effects on dopamine transporter activity. However, a strategy of how to antagonize drugs of abuse acting as substrate analogues is still elusive.     

Rapid Stereoselective Hydrolysis of (+)-Cocaine in Baboon Plasma Prevents Its Uptake in the Brain: Implications for Behavioral Studies

The naturally occurring enantiomer of cocaine, (–)-cocaine, has been previously labeled with ¹¹C on the N-methyl group and used in conjunction with positron emission tomography to show that cocaine is rapidly taken up in the striata of human and baboon brain. In the present study, the behaviorally inactive (+)-cocaine was similarly labeled, with a view to its use for measuring the nonspecific binding of cocaine. No brain uptake was seen, although transport of cocaine into the brain is not expected to be stereoselective. The explanation for the lack of uptake was determined to be very rapid metabolism of (+)-cocaine in the blood. By 30s after administration of labeled (+)-cocaine, it was undetectable in plasma. In vitro studies demonstrated that (+)-cocaine is 50% debenzoylated to (+)-ecgonine methyl ester within 5 s of exposure to baboon plasma but not to washed erythrocytes. The hydrolysis of (–)-cocaine is at least 1,000 times slower. Serum butyrylcholinesterase (EC 3.1.1.8) appears to be responsible for this hydrolysis, as evidenced by its inhibition by physostigmine and catalysis by commercially available pseudocholinesterase from horse and human blood.     

Characterization of a cocaine binding protein in human placenta

[3H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [3H]-cocaine with a high affinity site of 170 fmole/mg protein (Kd 16.7 nM). The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [3H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear.     

A lithium-induced conformational change in serotonin transporter alters cocaine binding, ion conductance, and reactivity of Cys-109.

Inactivation of serotonin transporter (SERT) expressed in HeLa cells by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) occurred much more readily when Na(+) in the reaction medium was replaced with Li(+). This did not result from a protective effect of Na(+) but rather from a Li(+)-specific increase in the reactivity of Cys-109 in the first external loop of the transporter. Li(+) alone of the alkali cations caused this increase in reactivity. Replacing Na(+) with N-methyl-d-glucamine (NMDG(+)) did not reduce the affinity of cocaine for SERT, as measured by displacement of a high affinity cocaine analog, but replacement of Na(+) with Li(+) led to a 2-fold increase in the K(D) for cocaine. The addition of either cocaine or serotonin (5-HT) protected SERT against MTSET inactivation. When SERT was expressed in Xenopus oocytes, inward currents were elicited by superfusing the cell with 5-HT (in the presence of Na(+)) or by replacing Na(+) with Li(+) but not NMDG(+). MTSET treatment of oocytes in Li(+) but not in Na(+) decreased both 5-HT and Li(+) induced currents, although 5-HT-induced currents were inhibited to a greater extent. Na(+) antagonized the effects of Li(+) on both inactivation and current. These results are consistent with Li(+) inducing a conformational change that exposes Cys-109, decreases cocaine affinity, and increases the uncoupled inward current.     

Cytotoxicity and genotoxicity of methyleugenol and related congeners-- a mechanism of activation for methyleugenol

Methyleugenol is a substituted alkenylbenzene found in a variety of foods, products, and essential oils. In a 2-year bioassay conducted by the National Toxicology Program, methyleugenol caused neoplastic lesions in the livers of Fischer 344 rats and B6C3F(1) mice. We were interested in the cytotoxicity and genotoxicity caused by methyleugenol and other alkenylbenzene compounds: safrole (a known hepatocarcinogen), eugenol, and isoeugenol. The endpoints were evaluated in cultured primary hepatocytes isolated from male Fischer 344 rats and female B6C3F(1) mice. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release, while genotoxicity was determined by using the unscheduled DNA synthesis (UDS) assay. Rat and mouse hepatocytes showed similar patterns of toxicity for each chemical tested. Methyleugenol and safrole were relatively non-cytotoxic, but caused UDS at concentrations between 10 and 500 microM. In contrast, isoeugenol and eugenol produced cytotoxicity in hepatocytes with LC50s of approximately 200-300 microM, but did not cause UDS. Concurrent incubation of 2000 microM cyclohexane oxide (CHO), an epoxide hydrolase competitor, with a non-cytotoxic concentration of methyleugenol (10 microM) resulted in increased cytotoxicity but had no effect on genotoxicity. However, incubation of 15 microM pentacholorophenol, a sulfotransferase inhibitor, with 10 uM methyleugenol resulted in increased cytotoxicity but had a significant reduction of genotoxicity. These results suggest that methyleugenol is similar to safrole in its ability to cause cytotoxicity and genotoxicity in rodents. It appears that the bioactivation of methyleugenol to a DNA reactive electrophile is mediated by a sulfotransferase in rodents, but epoxide formation is not responsible for the observed genotoxicity.     

Design of high-activity mutants of human butyrylcholinesterase against (-)-cocaine: structural and energetic factors affecting the catalytic efficiency

The present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (-)-cocaine by modeling the rate-determining transition state (TS1), i.e., the transition state for the first step of chemical reaction process, of (-)-cocaine hydrolysis catalyzed by various mutants of human BChE in comparison with the wild type. Molecular modeling of the TS1 structures revealed that mutations on certain nonactive site residues can indirectly affect the catalytic efficiency of the enzyme against (-)-cocaine through enhancing or weakening the overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. Computational insights and predictions were supported by the catalytic activity data obtained from wet experimental tests on the mutants of human BChE, including five new mutants reported for the first time. The BChE mutants with at least ～1000-fold improved catalytic efficiency against (-)-cocaine compared to the wild-type BChE are all associated with the TS1 structures having stronger overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. The combined computational and experimental data demonstrate a reasonable correlation relationship between the hydrogen-bonding distances in the TS1 structure and the catalytic efficiency of the enzyme against (-)-cocaine.     

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

Ethanol potentiates hypoxic liver injury: role of hepatocyte Na(+) overload

Centrilobular hypoxia has been suggested to contribute to hepatic damage caused by alcohol intoxication. However, the mechanisms involved are still poorly understood. We have investigated whether alterations of Na(+) homeostasis might account for ethanol-mediated increase in hepatocyte sensitivity to hypoxia. Addition of ethanol (100 mmol/l) to isolated rat hepatocytes incubated under nitrogen atmosphere greatly stimulated cell death. An increase in intracellular Na(+) levels preceded cell killing and Na(+) levels in hepatocytes exposed to the combination of ethanol and hypoxia were almost twice those in hypoxic cells without ethanol. Na(+) increase was also observed in hepatocytes incubated with ethanol in oxygenated buffer. Ethanol addition significantly lowered hepatocyte pH. Inhibiting ethanol and acetaldehyde oxidation with, respectively, 4-methylpyrazole and cyanamide prevented this effect. 4-methylpyrazole, cyanamide as well as hepatocyte incubation in a HCO(3)(-)-free buffer or in the presence of Na(+)/H(+) exchanger blocker 5-(N,N-dimethyl)-amiloride also reduced Na(+) influx in ethanol-treated hepatocytes. 4-methylpyrazole and cyanamide similarly prevented ethanol-stimulated Na(+) accumulation and hepatocyte killing during hypoxia. Moreover, ethanol-induced Na(+) influx caused cytotoxicity in hepatocytes pre-treated with Na(+), K(+)-ATPase inhibitor ouabain. Also in this condition 4-methylpyrazole and 5-(N,N-dimethyl)-amiloride decreased cell killing. These results indicate that ethanol can promotes cytotoxicity in hypoxic hepatocytes by enhancing Na(+) accumulation.     

Rat carboxylesterase ES-4 enzyme functions as a major hepatic neutral cholesteryl ester hydrolase

Although esterification of free cholesterol to cholesteryl ester in the liver is known to be catalyzed by the enzyme acyl-coenzyme A:cholesterol acyltransferase, ACAT, the neutral cholesteryl ester hydrolase (nCEH) that catalyzes the reverse reaction has remained elusive. Because cholesterol undergoes continuous cycling between free and esterified forms, the steady-state concentrations in the liver of the two species and their metabolic availability for pathways, such as lipoprotein assembly and bile acid synthesis, depend upon nCEH activity. On the basis of the general characteristics of the family of rat carboxylesterases, we hypothesized that one member, ES-4, was a promising candidate as a hepatic nCEH. Using under- and overexpression approaches, we provide multiple lines of evidence that establish ES-4 as a bona fide endogenous nCEH that can account for the majority of cholesteryl ester hydrolysis in transformed rat hepatic cells and primary rat hepatocytes.     

The isomers of cocaine and tropacocaine: Effect on 3H-catecholamine uptake by rat brain synaptosomes

Compared to (+)-$\text{pseudo}$cocaine, (?)-cocaine was 20 times more potent in inhibiting uptake of ³H-norepinephrine (³HNE) by cortical synaptosomes and 66 times more potent with respect to ³H-dopamine (³HDA) uptake by striatal synaptosomes. Although the tropacocaine isomers were equipotent as inhibitors of ³HNE uptake in the cortex, tropacocaine was 3.9 times more potent as an inhibitor of ³HDa uptake in the striatum than $\text{pseudo}$tropococaine. A major known cocaine metabolite, benzoylecgonine failed to inhibit the accumulation of ³HNE and ³HDA by synaptosomes from the cortex and striatum, respectively. The implications of these findings in relation to the motor stimulation seen with (?)-cocaine, (+)-$\text{pseudo}$cocaine and benzoylecgonine in rats are discussed.     

[3H]cocaine labels a binding site associated with the serotonin transporter in guinea pig brain: Allosteric modulation by paroxetine

We studied the characteristics of [³H]cocaine binding to membranes prepared from whole guinea pig brain. Cocaine binding was specific and saturable. A one-site binding model fit the data adequately: the Kd value of [³H]cocaine was 44 nM with a Bmax value of 280 fmol/mg protein. The rank order of potency for the [³H]cocaine binding site was paroxetine > clomipramine > (–)-cocaine > fluoxetine > mazindol > desipramine > GBR12909 > phencyclidine > benztropine > GBR12935 > (+)-cocaine. The IC50 values of these drugs for inhibition of [³H]cocaine binding were highly correlated with their IC50 values for inhibition of [³H]5-HT uptake into synaptosomes prepared from whole guinea pig brain. High affinity 5-HT uptake inhibitors produced dose-dependent wash-resistant (pseudoirreversible) inhibition of [³H]cocaine binding. The wash-resistant inhibition produced by paroxetine was due to an increase in the Kd of [³H]cocaine binding sites, and was accompanied by an increase in the dissociation rate, consistent with an allosteric mechanism. These studies suggest that, using membranes prepared from whole guinea pig brain, [³H]cocaine labels a binding site associated with serotonin transporter and that paroxetine and cocaine bind to different sites on the serotonin transporter.Abbreviations GBR12909 1-(2-{bis(4-fluorophenyl)methoxy}ethyl)-4-{3-phenylpropyl}piperazine - TCP 1-{1-(2-thienyl)cyclohexyl}piperidine - BTCP N-{1-(2-benzo(b)thiophenyl)cyclohexyl}piperidine - PCP 1-(1-phenylcyclohexyl)piperidine - GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) - CMI clomipramine     

Catalytic mechanism and energy barriers for butyrylcholinesterase-catalyzed hydrolysis of cocaine

The geometries of the transition states, intermediates, and prereactive enzyme-substrate complex and the corresponding energy barriers have been determined by performing hybrid quantum mechanical/molecular mechanical (QM/MM) calculations on butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)- and (+)-cocaine. The energy barriers were evaluated by performing QM/MM calculations with the QM method at the MP2/6-31+G* level and the MM method using the AMBER force field. These calculations allow us to account for the protein environmental effects on the transition states and energy barriers of these enzymatic reactions, showing remarkable effects of the protein environment on intermolecular hydrogen bonding (with an oxyanion hole), which is crucial for the transition state stabilization and, therefore, on the energy barriers. The calculated energy barriers are consistent with available experimental kinetic data. The highest barrier calculated for BChE-catalyzed hydrolysis of (-)- and (+)-cocaine is associated with the third reaction step, but the energy barrier calculated for the first step is close to the highest and is so sensitive to the protein environment that the first reaction step can be rate determining for (-)-cocaine hydrolysis catalyzed by a BChE mutant. The computational results provide valuable insights into future design of BChE mutants with a higher catalytic activity for (-)-cocaine.     

Aziridine biotransformation by microsomes and lethality to hepatocytes isolated from rat

To clarify the relationship of aziridine biotransformation to their cytotoxic activities, the metabolism of optical isomers of typical cytotoxic and non-cytotoxic aziridines was studied in isolated hepatocytes, rat liver microsomes, mitochondria and L-1210 mouse leukemia cells. Cytotoxic 1-methyl-2-beta-naphthylaziridine (NAZ) gave nitrosomethane as one of the bioactivation products in isolated hepatocytes and simultaneously induced a marked decrease in cellular ATP followed by cell lethality. NAZ itself did not directly affect the respiratory function of mitochondria in isolated hepatocytes or in buffer solution, however, it inhibited the mitochondrial activity in the presence of microsomes in the buffer solution. Nitroso-t-butane or nitrosomethane dimer, used as a substitute for extremely labile nitrosomethane, strongly inhibited the respiration of mitochondria. On the other hand, optical isomers of 2-aziridinecarboxylic acid (AZC) which did not give nitrosomethane in isolated hepatocytes or microsomes also did not show cytotoxicity. Thus, the cytotoxicity of NAZ seems to be induced by bioactivation via cellular oxidases with the nitrosomethane generated being a major toxic component. This may occur with most of the cytotoxic aziridine derivatives.     

A specific microassay for evaluating hepatic LDH activity in co-cultures of hepatocytes with other cells

This study describes the development of a simple, rapid and reproducible microassay for determining the intracellular LDH activity of rat hepatocytes present in a co-culture system with other cells. The procedure involves treatment of cellular homogenates with an anti-LDH antiserum that specifically inhibits the LDH activity of rat hepatocytes. The assay is performed in 96-well plates and LDH activity can be measured directly in the same wells using a colorimetric method. The difference in LDH activity values measured before and after antiserum incubation reflects the LDH content of the hepatocytes in the sample. The advantages of this method are the small number of cells required, a reduction in sample handling and the possibility of differentiating LDH activity in hepatic and non-hepatic cells. The possible applications of this technique as a parameter for biochemical data and as a test for cytotoxicity studies in co-cultures are also discussed.     

Modeling evolution of hydrogen bonding and stabilization of transition states in the process of cocaine hydrolysis catalyzed by human butyrylcholinesterase

Molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations were performed on the prereactive enzyme-substrate complex, transition states, intermediates, and product involved in the process of human butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)-cocaine. The computational results consistently reveal a unique role of the oxyanion hole (consisting of G116, G117, and A199) in BChE-catalyzed hydrolysis of cocaine, compared to acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine. During BChE-catalyzed hydrolysis of cocaine, only G117 has a hydrogen bond with the carbonyl oxygen (O31) of the cocaine benzoyl ester in the prereactive BChE-cocaine complex, and the NH groups of G117 and A199 are hydrogen-bonded with O31 of cocaine in all of the transition states and intermediates. Surprisingly, the NH hydrogen of G116 forms an unexpected hydrogen bond with the carboxyl group of E197 side chain and, therefore, is not available to form a hydrogen bond with O31 of cocaine in the acylation. The NH hydrogen of G116 is only partially available to form a weak hydrogen bond with O31 of cocaine in some structures involved in the deacylation. The change of the estimated hydrogen-bonding energy between the oxyanion hole and O31 of cocaine during the reaction process demonstrates how the protein environment can affect the energy barrier for each step of the BChE-catalyzed hydrolysis of cocaine. These insights concerning the effects of the oxyanion hole on the energy barriers provide valuable clues on how to rationally design BChE mutants with a higher catalytic activity for the hydrolysis of (-)-cocaine.     

Differential response of primary cultures of human and rat hepatocytes to aflatoxin B1-induced cytotoxicity and protection by the hepatoprotective agent (+)-cyanidanol-3

The acute hepatotoxicity induced by aflatoxin B1 (AFB1) and the potential protective effect of (+)-cyanidanol-3 (Catergen) were evaluated in both human and rat hepatocytes in primary cultures. AFB1-induced acute toxicity was visualized by light microscope observation and quantified by measurement of lactic dehydrogenase activity in the medium. Human hepatocytes were susceptible to AFB1-induced cytotoxicity but no evident relationship between the concentration of mycotoxin and the extent of cellular damage was established. (+)-Cyanidanol-3 was not toxic at concentrations up to 2 x 10(-3)M, but no obvious protective effect from AFB1-induced injury was evidenced in human cells. By contrast, rat hepatocytes responded in a dose-related manner to AFB1. (+)-Cyanidanol was toxic at 10(-3)M, but even at this concentration exerted a strong protective effect against AFB1-induced cytotoxicity. Such species differences suggest the existence of metabolic differences in both AFB1 and (+)-cyanidanol-3 activating and deactivating mechanisms.     

Contrasting role of Na(+) ions in modulating Cu(+2) or Cd(+2) induced hepatocyte toxicity

Previously we showed that hepatocyte lysis induced by Cu(+2)/Cd(+2) could be partly attributed to membrane lipid peroxidation induced by Cu(+2) or mitochondrial toxicity induced by Cd(+2) [5]. Changes in Na(+) and Ca(+2) homeostasis induced when Cu(+2) was incubated with hepatocytes markedly differed from that induced by Cd(+2). Na(+) omission from the media or addition of the Na(+)/H(+) exchange inhibitor 5-(N,N-dimethyl)-amiloride markedly increased Cu(+2) cytotoxicity even though Cu(+2) did not increase hepatocyte Na(+) when the media contained Na(+). Intracellular Ca(+2) levels however were markedly increased when the hepatocytes were incubated with Cu(+2) in a Na(+) free media and removing media Ca(+2) with EGTA also prevented Cu(+2) induced hepatocyte cytotoxicity. This suggests that intracellular Ca(+2) accumulation contributes to Cu(+2) induced cytotoxicity and a Na(+)-dependent Ca(+2) transporter is involved in controlling excessive Ca(+2) accumulation caused by Cu(+2). The omission of Cl(-) from the media or addition of glycine, a Cl(-) channel blocker also enhanced Cu induced cytotoxicity. By contrast Cd(+2) induced cytotoxicity was prevented by Na(+) omission from the media or by the addition of 5-(N,N-dimethyl)-amiloride. Furthermore the omission of Cl(-) from the media or addition of glycine also prevented Cd(+2) induced hepatocyte toxicity. A hypotonic media also increased Cd(+2) but not Cu(+2) induced hepatocyte cytotoxicity. This suggests that Cd(+2) but not Cu(+2) cytotoxicity could be partly attributed to disruption of cell volume regulation mechanisms. The increased osmotic load caused by the uncontrolled accumulation of intracellular Na(+) in Cd(+2) treated hepatocytes likely resulted from the activation of Na(+)/H(+) exchanger and the Na(+)/HCO(3)(-) cotransporter by the acidosis and ATP depletion caused by mitochondrial toxicity.     

Modulation of benzoquinone-induced cytotoxicity by diethyldithiocarbamate in isolated hepatocytes

The copper-chelating thiol drug diethyldithiocarbamate protected isolated hepatocytes from benzoquinone-induced alkylation cytotoxicity by reacting with benzoquinone and forming a conjugate which was identified by fast atom bombardment mass spectrometry as 2-(diethyldithiocarbamate-S-yl) hydroquinone. In contrast to benzoquinone, the conjugate was not cytotoxic to isolated hepatocytes. The thiol reductant dithiothreitol had no effect on benzoquinone-induced alkylation cytotoxicity. However, inactivation of catalase in the hepatocytes with azide and addition of the reducing agent ascorbate markedly enhanced the cytotoxicity of the conjugate but did not affect benzoquinone-induced cytotoxicity. Furthermore, inactivation of glutathione reductase and catalase in hepatocytes greatly enhanced the cytotoxicity of the conjugate and caused oxidation of GSH to GSSG. The conjugate also stimulated cyanide-resistant respiration, which suggests that the conjugate undergoes futile redox cycling resulting in the formation of hydrogen peroxide which causes cytotoxicity in isolated hepatocytes only if the peroxide detoxifying enzymes are inactivated. Diethyldithiocarbamate does, however, protect uncompromised isolated hepatocytes from benzoquinone cytotoxicity by conjugating benzoquinone, thereby preventing the electrophile from alkylating essential macromolecules. Diethyldithiocarbamate therefore changed the initiating cytotoxic mechanism of benzoquinone from alkylation to oxidative stress, which was less toxic.     

Cryopreservation of adult human hepatocytes obtained from resected liver biopsies

Isolated human hepatocytes have been shown to represent a valuable in vitro model to investigate the metabolism and cytotoxicity of xenobiotics. In addition, human hepatocyte transplantation and artificial liver support systems using isolated human hepatocytes are currently investigated as treatment for acute and chronic hepatic failure. In this regard, human hepatocyte banking by cryopreservation would be of great interest. In the present study, freshly isolated hepatocytes from resected liver biopsies of 28 separate donors (viability: 88 +/- 2%; plating efficiency: 79 +/- 5%) were cryopreserved using two different protocols, stepwise freezing (SF) or progressive freezing (PF), in combination (PF(+), SF(+)) or not (PF(-), SF(-)) with a 30 min preincubation in culture medium at 37 degrees C. Total recovery was higher after PF (38 +/- 3%) than after SF (12 +/- 2%). Preincubation prior to SF had no effect on plating efficiency of thawed hepatocytes (SF(-): 38 +/- 6% versus SF(+): 46 +/- 7%) while preincubation prior to PF increased plating efficiency of thawed hepatocytes (PF(-): 42 +/- 6% versus PF(+): 64 +/- 4%, p < 0.05). In attached cultured human cryopreserved/thawed hepatocytes (CH) from the PF(+) group, albumin production and glutathione content were not significantly different from those of the freshly isolated hepatocyte (FIH) cultures. Cells in CH monolayers appeared smaller than cells in FIH monolayers. In addition, the pattern of cytochrome P450- and UDP-glucuronosyl transferase-dependent isoenzyme activities and GST activity were different, suggesting a variability in the resistance to cryopreservation of the various liver hepatocyte populations. Taken all together, the results of the present study suggest that recovery of human hepatocytes after isolation prior to progressive freezing should allow human hepatocyte banking for use in pharmacotoxicology and cell therapy research purposes.