Fresh Liver Slices Research Articles

In Vitro Protective Effect of Ascorbic Acid Against Antibiotic-Induced Hepatotoxicity.

Although antibiotic-induced hepatotoxicity is recoverable with mild impairment, and some cases were reported to cause morbidity. However, an adjuvant is essential in reducing such incidences. The aim of this study is to evaluate the protective effect of ascorbic acid on antibiotic induced liver toxicity using liver slices. Fresh liver slices were incubated with different concentrations of sulfamethoxazole tetracycline and clavulanic acid along with ascorbic acid (200μg/ml) for 2 hours. The liver homogenate was assessed for markers like ALT, AST, MDA and CAT levels. Cytotoxicity assessment was performed using MTT assay. Incubating liver slices with all three antibiotics shows elevated levels of aminotransferases, MDA and CAT enzyme when compared to the control groups which indicates the level of hepatotoxicity. In the presence of ascorbic acid, the elevated levels of TBARS, ALT and AST were significantly reduced which showcases the protective effect of ascorbic acid. The percentage survival of cell was also shown to have improved while accessed using cell viability assay. Obtained data suggests that consuming vitamin C or vitamin C containing food like citrus fruits or green leafy vegetables equivalent to 3g/day during antibiotic treatment, perhaps put down the risk of liver toxicity to a greater extent.

Profile analysis of hepatic porcine and murine brain tissue slices obtained with a vibratome.

This study is aimed at characterizing soft tissue slices using a vibratome. In particular, the effect of two sectioning parameters (i.e., step size and sectioning speed) on resultant slice thickness was investigated for fresh porcine liver as well as for paraformaldehyde-fixed (PFA-fixed) and fresh murine brain. A simple framework for embedding, sectioning and imaging the slices was established to derive their thickness, which was evaluated through a purposely developed graphical user interface. Sectioning speed and step size had little effect on the thickness of fresh liver slices. Conversely, the thickness of PFA-fixed murine brain slices was found to be dependent on the step size, but not on the sectioning speed. In view of these results, fresh brain tissue was sliced varying the step size only, which was found to have a significant effect on resultant slice thickness. Although precision-cut slices (i.e., with regular thickness) were obtained for all the tissues, slice accuracy (defined as the match between the nominal step size chosen and the actual slice thickness obtained) was found to increase with tissue stiffness from fresh liver to PFA-fixed brain. This quantitative investigation can be very helpful for establishing the most suitable slicing setup for a given tissue.

Exploitation of the hepatic stellate cell Raman signature for their detection in native tissue samples.

Hepatic stellate cells (HSCs) surround liver sinusoids and store retinol while they are quiescent. During fibrotic liver diseases and acute-on-chronic liver failure they change to the activated state in which they proliferate, lose their retinol content and deposit extracellular matrix molecules. The process of HSC activation is of utmost interest, but so far only insufficiently understood, because there is a lack of techniques to address the function of single HSCs in the tissue context. In this contribution, the potential of Raman micro-spectroscopy for the label-free detection of HSCs in mouse liver samples is demonstrated. First, culture-induced activation of primary mouse HSCs is followed in vitro and characterized by means of Raman spectroscopy. The HSC activation state is confirmed by immunofluorescence labeling of glial fibrillary acidic protein (GFAP) and α-smooth muscle actin (ASMA). As expected, the unique Raman spectrum of retinol in quiescent HSCs is lost during activation. Nevertheless, successful discrimination of HSCs from primary hepatocytes is possible during all states of activation. A classification model based on principal component analysis followed by linear discriminant analysis (PCA-LDA) of the lipid droplet Raman data yields a prediction accuracy of 99%. The in vitro results are transferred to fresh liver slices and freshly sampled livers. Quiescent HSCs and a HSC transforming from quiescent to activated state are identified based on their Raman signature. This provides valuable information on HSC activation state in the liver.

Relationship Between Ischemia/Reperfusion Injury and the Stimulus of Fibrogenesis in an Experimental Model: Comparison Among Different Preservation Solutions

Background and Aims Orthotopic liver transplantation (OLT) has been the standard treatment for end-stage acute and chronic liver disease. Ischemia-reperfusion (I/R) injury is one of the major causes of poor graft function early after OLT, and adversely influencing graft and patient survivals. It is unknown whether I/R injury influences liver fibrogenesis. Materials and Methods Livers from 25 adult male Wistar rats were randomly assigned into 5 experimental groups according to the preservation solution: saline solution (SS); University of Wisconsin (UW) solution; Fructose 1, 6-biphosphate (FBP); S-Nitroso-N-Acetylcysteine (SNAC): or UW + SNAC (SNAC+UW). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactic dehydrogenase (LDH) were determined in preservation solution samples at 2, 4, and 6 hours. After 6 hours of cold ischemia, ex situ reperfusion was applied to the liver for 15 minutes. Serum AST, ALT, LDH, and renin levels were determined. Fresh liver slices were processed for histological studies, determination of thiobarbituric acid reactive substances, catalase, and glutathione, and expression of TGF-β1 and angiotensin II AT1 receptor. Results AST was significantly lower during cold storage with UW than with the older media ( P = .001); ALT was lower in the FBP group ( P = .023) and LDH was lower in the FBP and SNAC groups ( P = .007). After reperfusion, serum AST, ALT, LDH, and TBARS showed no significant differences among the groups. Catalase was significantly lower in the SS and FBP groups ( P = .008 and P = .006, respectively). Compared with UW, glutathione concentrations were significantly higher in SS, FBP, and SNAC 200 ( P = .004). Renin levels were significantly lower in the FBP group ( P = .022). No histological signs of preservation injury were observed in the hepatic sample. No expressions were detected of TGF-β1 or AT1 receptor. Conclusion In this experimental model of early reperfusion injury, preservation changes related to higher levels of renin, which suggest its role in fibrogenesis. FBP was associated with lower renin levels than other solutions including UW.

Inhibition of Hepatitis C Virus Replication by Antimonial Compounds

Chronic hepatitis C virus (HCV) infection is a worldwide health problem causing serious complications, such as liver cirrhosis and hepatoma. Alpha interferon (IFN-alpha) or its polyethylene glycol-modified form combined with ribavirin is the only recommended therapy. However, an alternative therapy is needed due to the unsatisfactory cure rate of the IFN-based therapy. Using a modified reporter assay based on the HCV subgenomic-replicon system, we found that sodium stibogluconate (SSG), a compound used for leishmania treatment, suppressed HCV replication. We have previously reported that SSG is effective at inhibiting HCV replication in a cell line permissive for HCV infection/replication and in an ex vivo assay using fresh human liver slices obtained from patients infected with HCV (26). In this study, we show that the SSG 50% inhibitory dose for HCV replication is 0.2 to 0.3 mg/ml (equivalent to 345 to 517 microM of Sb) in the HCV subgenomic-replicon system. We also found that SSG and IFN-alpha exert a strong synergistic anti-HCV effect in both the traditional isobologram analysis and the median effect principle (CalcuSyn analysis). The combination of SSG and IFN-alpha could sustain the antiviral response better than SSG or IFN-alpha alone. The results suggest that SSG may be a good drug candidate for use in combination with other therapeutics, such as IFN-alpha and ribavirin, to treat HCV infection.

Metabolism of 2-phenylethylamine and phenylacetaldehyde by precision-cut guinea pig fresh liver slices.

2-Phenylethylamine is an endogenous constituent of human brain and is implicated in cerebral transmission. It is also found in certain foodstuffs and may cause toxic side-effects in susceptible individuals. Metabolism of 2-phenylethylamine to phenylacetaldehyde is catalyzed by monoamine oxidase and the oxidation of the reactive aldehyde to its acid derivative is catalyzed mainly by aldehyde dehydrogenase and perhaps aldehyde oxidase, with xanthine oxidase having minimal transformation. The present investigation examines the metabolism of 2-phenylethylamine to phenylacetaldehyde in liver slices and compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity in the oxidation of phenylacetaldehyde with precision-cut fresh liver slices in the presence/absence of specific inhibitors of each enzyme. In liver slices, phenylacetaldehyde was rapidly converted to phenylacetic acid. Phenylacetic acid was the main metabolite of 2-phenylethylamine, via the intermediate phenylacetaldehyde. Phenylacetic acid formation was completely inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent and allopurinol (specific inhibitor of xanthine oxidase) had little or no effect. Therefore, in liver slices, phenylacetaldehyde is rapidly oxidized by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.

Inhibition of authentic hepatitis C virus replication by sodium stibogluconate

Using a hepatitis C virus (HCV) subgenomic RNA replicon system, drugs currently being used to treat other human diseases were examined for their antiviral activities against HCV. Several drugs including sodium stibogluconate, a compound used to treat leishmaniasis, were capable of suppressing replication of HCV replicon. The antiviral effect of sodium stibogluconate was subsequently verified using a cell line (293EBNA-Sip-L) previously proved to be permissive for HCV infection/replication. An ex vivo assay using fresh human liver slices established and a panel of human liver slices was obtained from biopsy samples of patients infected with HCV was used to examine the antiviral activity of this drug. A nearly complete suppression effect was achieved in four of six human liver slices at the drug concentration of 100 μg/ml, lower than what was required to treat leishmaniasis. A human trial is mandatory to understand its clinical value in treating chronic hepatitis C.

Rates of metabolism of chlorzoxazone, dextromethorphan, 7-ethoxycoumarin, imipramine, quinidine, testosterone and verapamil by fresh and cryopreserved rat liver slices, and some comparisons with microsomes

In the present study we have investigated the disappearance of chlorzoxazone, dextromethorphan, 7-ethoxycoumarin, imipramine, quinidine, testosterone and verapamil from the medium in which fresh and cryopreserved rat liver slices were incubated. These compounds are all substrates of major isoforms of cytochrome P450 expressed in the liver. The metabolism of five of these compounds in microsomes from rat liver was also examined. Determinations of the concentrations of the compounds were performed employing LC/MS. Intrinsic clearance values (CL ints) were calculated on the basis of the concentration–vs.–time curves. No significant differences in the CL int values obtained with fresh and cryopreserved rat liver slices were observed for any of the compounds. The highest CL int value estimated with liver slices was observed for testosterone and the lowest values were with chlorzoxazone and 7-ethoxycoumarin. The total CL int values for 7-ethoxycoumarin and imipramine, calculated using scaling factors, were similar for liver slices and microsomes. In the case of testosterone, this total CL int was approximately 3.7-fold lower, whereas for dextromethorphan and quinidine it was 2.5- and 8.5-fold higher, respectively, with liver slices than with microromes. In conclusion, the rate of metabolism of the seven compounds tested with rat liver slices was not affected by cryopreservation. This finding adds further support to the general conclusion that the major activities involved in drug metabolism are not affected by cryopreservation of rat liver slices.

Glucuronidation of 4-methylumbelliferone and 4-hydroxybiphenyl and in vitro induction of UDP-glucuronosyltransferase 2B12-mRNA in precision-cut rat liver slices

Fresh rat liver slices were used to demonstrate the glucuronidation of the model substrates 4-methylumbelliferone (MU) and 4-hydroxybiphenyl (HB). Both glucuronidation reactions proved to be more stable than cytochrome P450-dependent monooxygenations. After an incubation time of 48 h there was no decrease in MU glucuronidation rate, whereas HB glucuronidation was stable until 24 h, and then decreased by about 50% until 48 h. The technique of quantitative competitive RT-PCR was used to determine the expression of UDP-glucuronosyltransferase 2B12-mRNA (UGT2B12-mRNA) in precision-cut rat liver slices. Constitutive levels of UGT2B12-mRNA were measurable. Following 24 h culture of rat liver slices in the presence of phenobarbital, the level of UGT2B12-mRNA increased about twofold, which corresponds to the inducibility in vivo. The addition of beta-naphthoflavone had no influence. The results show that precision-cut liver slices are not only suitable for the detection of an in vitro induction of cytochrome P450-mRNAs, which is characterized by high induction factors, but also of poor induction effects, e.g. on UGT2B12-mRNA, provided that the respective mRNA is exactly quantified.

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 μM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 μM pregnenolone 16α-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.

Effects of di-isononyl phthalate, di-2-ethylhexyl phthalate, and clofibrate in cynomolgus monkeys.

The effects of the peroxisome proliferators di-isononyl phthalate (DINP) and di-2-ethylhexyl phthalate (DEHP) were evaluated in young adult male cynomolgus monkeys after 14 days of treatment, with emphasis on detecting hepatic and other effects seen in rats and mice after treatment with high doses of phthalates. Groups of 4 monkeys received DINP (500 mg/kg/day), DEHP (500 mg/kg/day), or vehicle (0.5% methyl cellulose, 10 ml/kg) by intragastric intubation for 14 consecutive days. Clofibrate (250 mg/kg/day), a hypolipidemic drug used for cholesterol reduction in human patients was used as a reference substance. None of the test substances had any effect on body weight or liver weights. Histopathological examination of tissues from these animals revealed no distinctive treatment-related effects in the liver, kidney, or testes. There were also no changes in any of the hepatic markers for peroxisomal proliferation, including peroxisomal beta-oxidation (PBOX) or replicative DNA synthesis. Additionally, in situ dye transfer studies using fresh liver slices revealed that DINP, DEHP, and clofibrate had no effect on gap junctional intercellular communication (GJIC). None of the test substances produced any toxicologically important changes in urinalysis, hematology, or clinical chemistry; however, clofibrate produced some emesis, small increases in serum triglyceride, decreased calcium, and decreased weights of testes/epididymides and thyroid/parathyroid. The toxicological significance of these small changes is questionable. The absence of observable hepatic effects in monkeys at doses that produce hepatic effects in rodents suggests that DINP, DEHP, and clofibrate would also not elicit in primates other effects such as liver cancer. These data, along with results from in vitro hepatocyte studies, indicate that rodents are not good animal models for predicting the hepatic effects of phthalates in primates, including humans.

Use of Fresh and Cryopreserved Human Liver Slices in Toxicology with Special Reference to In vitro Induction of Cytochrome P450

Human liver slices were prepared with a Krumdieck slicer from macroscopically healthy surgical waste after partial hepatectomy. They were incubated without or with the addition of the inducer β-naphthoflavone (BNF) (25 μ m) either immediately after preparation (fresh slices) for up to 24 hours or after cryopreservation in liquid nitrogen (thawed slices) for up to 6 hours. Potassium concentration was well maintained in fresh and thawed slices over 24 and 6 hours, respectively, but at lower levels than in rat liver slices. Albumin secretion showed relatively large interindividual differences. Both parameters were lower in thawed slices than in fresh ones, but indicated a certain number of viable cells. In untreated fresh slices CYP1A1-mRNA was not detectable; however, it increased distinctly within 6 hours of exposure to BNF. The amounts of induced CYP1A1-mRNA differed by a factor of more than 100 among six human livers and were lower than in fresh rat liver slices. Even in thawed human slices, CYP1A1-mRNA expression could be induced in vitro by BNF, although at a very low level and preferentially in those specimens with comparably high inducibility already before freezing.

Expression of cytosolic beta-glucosidase in guinea pig liver cells.

The cytosolic beta-glucosidase of mammalian liver has been implicated in the metabolic transformation of plant glycosides, such as vicine and amygdalin, which are associated with the development of toxic syndromes. We investigated which cell types express cytosolic beta-glucosidase in guinea pig liver, and characterized the contribution of this enzyme to the hydrolysis of aromatic glucosides in cultured cells and in tissue slices. Cytosolic beta-glucosidase was expressed in hepatocytes and not in Kupffer or endothelial cells as determined by enzyme-specific activity and Western blots of liver cell extracts. Intracellular beta-glucosidase activity was visualized using the fluorescent beta-glucosidase substrate, resorufin beta-D-glucoside, and shown to be caused by the cytosolic beta-glucosidase using the inhibitors, conduritol beta-epoxide and dinitrophenol-2-deoxy-2-fluoro-beta-D-glucopyranoside (DNP2FGlc). Staining of fresh liver slices with resorufin beta-glucoside revealed that cytosolic beta-glucosidase is expressed in all hepatocytes, with no significant portal-central gradient. These data indicate that cytosolic beta-glucosidase is a hepatocyte-specific enzyme, and support the hypothesis that cytosolic beta-glucosidase in the liver functions to hydrolyze small glucosides absorbed by the intestine. Furthermore, toxic injury to cultured hepatocytes by CCl4 resulted in release of cytosolic beta-glucosidase in parallel with the hepatocyte marker enzymes alanine transaminase and lactate dehydrogenase. This suggests that acute increases in serum levels of cytosolic beta-glucosidase in animal models of liver injury may reflect direct injury of hepatocytes.

A rapid and simple method for cryopreservation of human liver slices

1. Precision-cut liver slices represent a suitable and convenient in vitro preparation for studying metabolism and toxicity mechanisms of drugs and toxic chemicals.Particularly in the case of human liver slices, cryopreservation would enable more efficient utilization of this scarce and irregularly available tissue. 2. Liver slices from consecutive human livers were cryopreserved using a method previously developed for rat and monkey liver slices. This procedure involves incubation in 12% dimethyl sulphoxide for 30 min on ice and direct immersion into liquid nitrogen. 3. Functional integrity of cryopreserved human liver slices, as compared with that of fresh liver slices, was maintained at 66±8% (alanine aminotransferase activity retained in the slices), 78±7% (urea synthesis), 88±14% (testosterone hydroxylation), 84±7% (N-deethylation of lidocaine) and 88±10% (total O-deethylation of 7-ethoxycoumarin). The ratios of testosterone metabolites did not change on cryopreservation. 4. These results show that the cryopreserved human liver slices retained the measured drug metabolism activities. Therefore, this cryopreservation method is suitable for storing liver slices to be used for comparing drug metabolism patterns, at least qualitatively, between species.

Estrogen Up-regulates Apolipoprotein E (ApoE) Gene Expression by Increasing ApoE mRNA in the Translating Pool via the Estrogen Receptor α-Mediated Pathway

The antiatherogenic property of estrogens is mediated via at least two mechanisms: first by affecting plasma lipoprotein profiles, and second by affecting the components of the vessel wall. Raising plasma apolipoprotein E (apoE) in mice protects them against diet-induced atherosclerosis (Shimano, H., Yamada, N., Katsuki, M., Gotoda, T., Harada, K., Murase, T., Fukuzawa, C., Takaku, F., and Yazaka, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1750-1754). It is possible that estrogen may be antiatherogenic at least in part by increasing plasma apoE levels. Therefore, we studied the regulation of apoE by estrogen. A survey of 15 inbred strains of mice showed that some mouse strains responded to injections or subcutaneously implanted pellets of estradiol by raising their apoB and apoE levels and some did not. We performed detailed studies in two "responder" strains, C57L and C57BL, and two "non-responder" strains, C3H and BALBc. Responders increased their plasma apoE levels 2.5-fold. Non-responders' levels were altered +/-10%. In the responders the distribution of apoE among the plasma lipoproteins shifted from high density lipoprotein toward the apoB-containing lipoprotein fractions. In nonresponders the shift was toward high density lipoprotein. Hepatic apoE mRNA levels and relative rates of apoE mRNA transcription were unchanged in all strains, suggesting that apoE regulation occurred at posttranscriptional loci. Therefore, we measured apoE synthesis in fresh liver slices and on isolated hepatic polysomes. Two-fold increases were noted but only in responders accompanied by selective 1.5-fold increases in polysomal apoE mRNA levels. Similar increases in apoE synthesis were also observed in castrated C57BL mice given either physiological or pharmacological replacement doses of estradiol, but not testosterone, suggesting that the effect of estradiol was specific on the distribution of apoE mRNA in the translationally active polysomal pool. Next, we examined whether the effects of estrogen on apoE translation were mediated by estrogen receptors (ER). ER-alpha knock-out mice and their wild-type littermates were administered estradiol. As expected, apoE levels and hepatic apoE synthesis increased more than 2-fold in the wild-type littermates, but only 20% increases in the plasma apoE and hepatic synthesis were observed in the ER knock-out mice. Hepatic apoE mRNA levels did not change in either the wild-type or the ER knock-out mice. Thus, estradiol up-regulates apoE gene expression by increasing levels of apoE mRNA in the polysomal translating pool. Furthermore, the increased polysomal recruitment of apoE mRNA is largely mediated by estrogen receptors.

In vitro glucuronidation of D-23129, a new anticonvulsant, by human liver microsomes and liver slices

1. The metabolic profile of D-23129, a new anticonvulsant agent, was studied in vitro using human liver microsomes and fresh liver slices. 2. Oxidative metabolism appeared to be minimal with D-23129. The percent mean total radioactivity not associated with the parent compound recovered from oxidative metabolism studies from three individual liver donors was 0 7% 0 6 SD and was not significantly different from \\ [C]-D-23129 incubated with heat inactivated microsomes, mean 0 5% 0 4 SD. 3. Phase II conjugation dominated the metabolism of D-23129 producing two distinct N -glucuronides as the primary metabolites. These metabolites were identified by electrospray ionization LC MS. 4. The apparent K for one of the glucuronide metabolites was determined in human m liver microsome preparations from two individual liver donors to be 131 and 264 mu M respectively. V determined for the same microsomal preparations yielded 48 9 and max 59 9 pmol min mg protein.

Pharmacokinetics and oral bioavailability of exogenous melatonin in preclinical animal models and clinical implications.

A review of the literature indicates that the absolute oral bioavailability of exogenous melatonin in humans or in preclinical animal models has not been adequately characterized; hence, this study was undertaken. Pharmacokinetics of melatonin was studied in rats, dogs, and monkeys following intravenous and oral administrations, and the absolute oral bioavailability of melatonin was calculated from the area under the plasma concentration-time curve. The apparent elimination half-life of melatonin following an intravenous dose of 3 mg/kg (5 mg/kg in rats) was 19.8, 18.6, and 34.2 minutes, respectively, in rats, dogs, and monkeys. The dose normalized oral bioavailability of melatonin following a 10 mg/kg oral dose was 53.5% in rats, while it was in excess of 100% in dogs and monkeys. Further, bioavailability of melatonin following a 10 mg/kg intraperitoneal administration in rats was 74.0%, suggesting the lack of substantial first-pass hepatic extraction of melatonin in rats. However, the oral bioavailability of melatonin in dogs decreased to 16.9% following a 1 mg/kg oral dose, indicating dose-dependent bioavailability in dogs. In vitro permeability studies with CACO-2 cells suggest that melatonin is likely to be well absorbed in humans. In vitro metabolism studies with fresh liver slices from rats as well as human donors were conducted to compare the initial rates of metabolism of melatonin between the two species and the results suggest that the intrinsic clearance of melatonin in humans may be lower than that in rats.

Absence of change in hepatic lactate metabolism after burn injury

In response to severe injury, extraordinary quantities of lactate that are released from the peripheral tissues serve as substrate for hepatic gluconeogenesis. It is possible that as a result of burn injury, reaction kinetics involving lactate could be directly changed within the liver. The metabolic fate of [U- 14C]lactate was examined in vitro in fresh liver slices after a 20 per cent total body surface area injury. Glucose and CO 2 were produced in vitro by the liver tissues after the injury and no differences were seen in the metabolism of these substrates between the injured and control animals. These findings suggest that the intrinsic enzymatic processes within the liver are not directly altered by injury itself or by any of the associated inflammatory mediators which appear early after burns.

Biotransformation activity in vitrified human liver slices

In vitro testing of human liver for biotransformation or xenobiotic metabolism studies has been limited by unpredictable acquisition of samples. Consequently, it has become necessary to consider methods to cryopreserve and store these samples whenever they do become available for culture of the revived tissue at a more convenient time. Human liver slices were cryopreserved by vitrification, which allows for the transfer of aqueous media to low temperatures (−196 °C) without the formation of ice crystals. Human liver slices were exposed to increasing concentrations of 1,2-propanediol up to a final concentration of 4.76 M in fetal calf serum. Slices were then vitrified by direct immersion into liquid nitrogen and warmed by submersion in 37 °C fetal calf serum. Warming was done either immediately or after 4 and 8 weeks of storage under liquid nitrogen. The effects of vitrification, storage time, and warming on biotransformation were determined by assessing the integrated metabolism of 7-ethoxycoumarin (7-EC). Vitrified or fresh human liver slices were exposed to 50 μ M 7-EC and its primary metabolite 7-hydroxycoumarin (7-HC) in organ culture for up to 6 hr. Metabolite production of both fresh and vitrified liver slices was compared. Retention of the inherent biotransformation rate was usually high and seemed independent of storage time. Integration of both cytochrome P450-mediated and secondary conjugation processes was retained in cryopreserved tissue. Vitrification offers a way to cryopreserve human liver slices for the study of xenobiotic metabolism in humans.

Adult rat liver slices as a model for studying the hepatotoxicity of vincaalkaloids

There are certain disadvantages associated with the use of isolated or cultured cells including the need to use proteolytic enzymes for their isolation and loss of tissue organization. In order to provide an in vitro system for toxicological studies that preserves tissue integrity, a method for preparation and incubation of adult rat liver slices has been developed. Fresh ultra-thin liver slices were produced in large quantities at a rapid rate under conditions that cause minimal tissue trauma. They were incubated in a system that has been designed to allow optimum gas and nutrient diffusion. Several biochemical parameters (lactate dehydrogenase (LDH) leakage, ATP content, protein synthesis and secretion) were monitored during a 20-hr incubation period. This liver slice model was used to study the toxicity of four vincaalkaloids: vincristine, vindesine, vinblastine and navelbine. Treatment with the vincaalkaloids resulted in an inhibition of protein synthesis and secretion without any effect on LDH leakage.