Galactosamine hepatotoxicity: effect of galactosamine on glutathione resynthesis in rat primary hepatocyte cultures.

The effect of galactosamine on the resynthesis of glutathione in rat primary hepatocyte cultures was investigated. Cultured rat hepatocytes were treated with galactosamine (4 mM) 1.5 hr prior to concurrent with, or 1.5 hr after cell attachment; total cellular glutathione was then measured over time. Addition of galactosamine at any of these times suppressed methionine-enhanced glutathione resynthesis in the cultures after a lag period of about 120 min. The lag period was not due to slow uptake of galactosamine by the cultured cells, since cellular UTP levels fell to less than 10% of controls within 60 min, a time frame comparable to that observed in vivo. Neither was the lag period a result of interference with cellular uptake of methionine or with conversion of methionine to cysteine, since the phenomenon was observed regardless of whether methionine or cysteine was used to promote glutathione resynthesis. Addition of uridine, which protects against galactosamine hepatotoxicity in vivo by replenishing hepatic UTP levels, did not prevent the suppression of glutathione resynthesis. The data indicate that (a) galactosamine inhibits the time-dependent resynthesis of glutathione in primary hepatocyte cultures, (b) a lag period exists for this response, and (c) this effect is not directly related to depletion of cellular UTP stores.     

Lobar variation of carbon tetrachloride hepatotoxicity in the rat

The lobar variation m carbon tetrachloride-induced liver damage in the rat has been assessed by use of a systematic random sampling protocol and quantitative morphometry. A random inter-animal lobar variation in severity of damage was apparent, in contrast with the results obtained previously in which sampling bias, small animal numbers, and lack of fully quantitative measurement were apparent. The route of administration (oral vs. i.p.) did not influence these findings.     

Human hepatocyte cultures: a model of pharmacotoxicological studies

1. Viable adult human hepatocytes were obtained in large yields by perfusion of the liver of kidney donors. The hepatocytes were cultured either alone or in association with rat-liver epithelial cells.

2. In pure culture the survival of hepatocytes did not exceed two to three weeks, while in co-culture they survived for several weeks and better retained the specific liver functions of albumin secretion, cytochrome P-450 content and glucuronidation of drugs.

3. Human hepatocytes, particularly when mixed with rat-liver epithelial cells, may provide a valuable tool for predicting the metabolic pathways and hepatotoxicity of new-drugs in man.     

Human hepatocyte cultures: a model of pharmaco-toxicological studies

Viable adult human hepatocytes were obtained in large yields by perfusion of the liver of kidney donors. The hepatocytes were cultured either alone or in association with rat-liver epithelial cells. In pure culture the survival of hepatocytes did not exceed two to three weeks, while in co-culture they survived for several weeks and better retained the specific liver functions of albumin secretion, cytochrome P-450 content and glucuronidation of drugs. Human hepatocytes, particularly when mixed with rat-liver epithelial cells, may provide a valuable tool for predicting the metabolic pathways and hepatotoxicity of new drugs in man.     

Mechanistic studies on carbon tetrachloride hepatotoxicity in fasted and fed rats

In agreement with previous reports from other laboratories, the fed rats are more resistant to the toxic action of CCl₄ than rats starved overnight. This preventive effect of feeding does not merely reflect a delay in the time for damage to appear, since the protection is even more pronounced at 72 than at 24 hr. The similarity in CCl₄ concentrations in the livers of fed and fasted rats excludes an effect due to a decreased absorption of the hepatotoxin. Irreversible binding of ¹⁴CCl₄ to liver microsomal lipids and lipid peroxidation are only slightly decreased in the livers of fed rats when compared to that of fasted rats. These findings correlate with the similarity of the sleeping time, cytochrome P-450 content and cytochrome P-650 reductase activity in both groups. Preventive effects are apparently due to an interference in the events occurring between CCl₄ activation or lipid peroxidation and the resulting damage.     

Utilization of methionine as a sulfhydryl source for metallothionein synthesis in rat primary hepatocyte cultures

Metallothioneins (MT) contain a high concentration of cysteine which bind heavy metals. Exposure of liver cells to metals induces the synthesis of MT and thus causes the cells to draw upon their sulfhydryl (SH) pools. The utilization of methionine as compared with that of cysteine as a source of SH for the synthesis of MT has not been shown. Therefore, studies were designed to determine whether methionine, in addition to cysteine, serves as an SH donor for Zn-induced synthesis of MT in rat hepatocyte cultures. Hepatocytes were able to synthesize only low levels of MT when the concentration of both amino acids was extremely low; however, when either of the amino acids was present at a high concentration, production of MT was independent of the other amino acid concentration. Subsequently, induction of MT was compared in four media: complete (0.5 mM methionine, 0.5 mM cysteine), Met (0.5 mM methionine), Cys (0.5 mM cysteine), and SH free (-SH). Somewhat higher concentrations of MT were produced by the hepatocytes in the Met than in the Cys media and no differences were observed between the Met and the complete media. By contrast, GSH synthesis was much more dependent on methionine than on cysteine for its synthesis. Incorporation studies with 35S-labeled cysteine and methionine indicated that lower concentrations of MT found in hepatocytes in the Cys media may be due to less accumulation of cysteine by the hepatocytes. Cellular accumulation of cysteine was initially rapid and then reached a plateau, whereas the rate for methionine accumulation was more constant and eventually obtained higher cellular levels. To provide additional evidence for the role of methionine in MT production, a known inhibitor of the cystathionine pathway, DL-propargylglycine (PPG), was added to each of the four media. Reductions in MT levels were not observed in the cells cultured in the complete and Cys media; however, a 95% reduction was observed in the cells cultured in the Met media. In summary, the present results suggest that both cysteine and methionine can serve as a SH source for MT synthesis, and that the availability of SH in most culture mediums would not limit the synthesis of MT. Whereas methionine is a much better SH source than cysteine for GSH synthesis in hepatocyte cultures, it is only slightly better for MT synthesis.     

Bovine corneal epithelial primary cultures as an in vitro model for ophthalmic drugs studies

In the recent years a significant development of investigations with regard to bioavailability of ocular drugs has been noticed. The corneal epithelial barrier is the main pathway for ocular penetration of topically applied ophthalmic drugs into the anterior chamber. To work out an in vitro model of bovine corneal epithelial primary cultures and exercise it for permeability research with lipophilic and hydrophilic markers, permeability coefficients estimation of the 6-carboxyfluorescein and rhodamin B was made. The corneal epithelial cultures of the 3th or 4th passage were chosen for layered culture with inserts based on the liquid-liquid interface (for the first week) and the air-liquid interface (for the two following weeks). On the 7th, 12th, 18th, 21st experiment day TER values, and on the 21st day drug permeability coefficients, were determined. The mean TER values of the 7th, 12th, 18th, 21st day of corneal epithelial culture were: 122.14, 155.14, 198.43 and; 247.43 Wcm2, respectively. The mean values of permeability coefficients on the 21st day of culture for 6-carboxyfluorescein and rhodamin B were 3.87 +/-0.10 x 10(-6)cm/s and 3.65 +/- 0.06 x 10(-6)cm/s, respectively. We state that the in vitro bovine corneal epithelial primary culture model is useful for ocular studies.     

Prevention and treatment of carbon tetrachloride hepatotoxicity by cysteine: studies about its mechanism

Cysteine administration to rats (1.9 g/kg po) prevented the development of the necrosis and fatty liver induced by CCl₄. This protective effect was observed when cysteine was given either 30 min before or 1 hr after the administration of CCl₄. Cysteine administration did not prevent the irreversible binding of ¹⁴C from ¹⁴CCl₄ to microsomal lipids, but it partially reduced the binding to microsomal proteins at 6 hr after ¹⁴CCl₄. Cysteine pretreatment did not modify the intensity of the CCl₄-induced lipid peroxidation process or cytochrome P-450 destruction, but it partially prevented depression of glucose-6-phosphatase activity by CCl₄. The results are compatible with the possibility of a protective action of cysteine at a site in the chain of events leading to necrosis, but not the activation step.     

Ethanol potentiation of carbon tetrachloride hepatotoxicity: possible role for the in vivo inhibition of aldehyde dehydrogenase

A potentiation of CCl4-induced hepatotoxicity was observed in rats pretreated with ethanol 18 hr prior to CCl4 exposure. Hepatic microsomal aldehyde dehydrogenase (ALDH) was significantly inhibited in animals sacrificed 1 hr following the sequential exposure, however, no more so than in those animals receiving CCl4 alone. The animals receiving ethanol alone had ALDH activity similar to vehicle treated controls. Twenty-four hours following a potentiating dose of ethanol and CCl4 an 81 and 57% decline in NAD+-dependent microsomal and mitochondrial ALDH activity was observed, respectively. Similar results were observed for microsomal and mitochondrial NADP+-dependent ALDH activity. The decline in membrane-bound ALDH was greater in potentiated animals than in those receiving CCl4 alone. A relatively smaller decline in cytosolic ALDH activity was observed in CCl4 treated rats with or without ethanol pre-exposure. The data suggest that inhibition of membrane bound ALDH may be one of the major mechanisms of in vivo potentiation of CCl4-induced hepatotoxicity by ethanol.     

Biochemical mechanisms and morphological selectivity in hepatotoxicity: studies in cultures of hepatic-parenchymal and non-parenchymal cells

Primary cultures of rat-liver parenchymal and non-parenchymal cells have been used to study some of the factors influencing the selective injury that can be caused in vivo by the direct-acting hepatotoxins beryllium, cadmium, ricin and modeccin to either liver-parenchymal or non-parenchymal cells. The studies on beryllium and cadmium compounds show that it is necessary to consider the chemical species generated in the culture medium, since particulate or colloidal forms are taken up predominantly by non-parenchymal cells whereas soluble forms more readily enter parenchymal cells. The studies with the glycoproteins ricin and modeccin illustrate the importance in their selective cell toxicity of specific membrane-recognition processes present in liver cells, particularly uptake in non-parenchymal cells through interactions with terminal mannose oligosaccharides in the toxins.     

Biochemical mechanisms and morphological selectivity in hepatotoxicity: studies in cultures of hepatic-parenchymal and non-parenchymal cells

1. Primary cultures of rat-liver parenchymal and non-parenchymal cells have been used to study some of the factors influencing the selective injury that can be caused in vivo by the direct-acting hepatotoxins beryllium, cadmium, ricin and modeccin to either liver-parenchymal or non-parenchymal cells.

2. The studies on beryllium and cadmium compounds show that it is necessary to consider the chemical species generated in the culture medium, since particulate or colloidal forms are taken up predominantly by non-parenchymal cells whereas soluble forms more readily enter parenchymal cells.

3. The studies with the glycoproteins ricin and modeccin illustrate the importance in their selective cell toxicity of specific membrane-recognition processes present in liver cells, particularly uptake in non-parenchymal cells through interactioiis with terminal mannose oligosaccharides in the toxins.     

Induction of metallothionein in rat primary hepatocyte cultures: evidence for direct and indirect induction

The ability of a number of metals and organic chemicals to induce metallothionein (MT) synthesis in primary cultures of rat hepatocytes was tested to determine whether MT induction in vivo results from a direct effect of the agent on the liver or as a result of an indirect, physiologic response to the agent. Hepatocytes were exposed to metals [zinc (Zn), cadmium (Cd), mercury (Hg), manganese (Mn), lead (Pb), cobalt (Co), nickel (Ni), and vanadium (V)] or organic compounds [ethanol, urethane, L-2-oxothiozolidine 4-carboxylate (L-OTCA), or dexamethasone] and were assayed for metallothionein by the Cd/hemoglobin radioassay. Cell viability was monitored by protein synthesis activity and cellular K+ concentration. Increases in MT concentrations were noted for Zn (22-fold), Hg (6.4-fold), Cd (4.8-fold), Co (2.4-fold), Ni (2.2-fold), and dexamethasone (4.5-fold). However, even at maximum tolerated concentrations, Mn, Pb, V, ethanol, urethane, and L-OTCA did not increase MT. The results indicate that Zn, Cd, Hg, Co, Ni and dexamethasone induce MT in vitro and thus are direct inducers of MT synthesis in hepatic tissue. In contrast, Mn, Pb, ethanol, urethane and L-OTCA, which did not increase the MT content of hepatocytes, apparently do so in vivo by an indirect mechanism.     

Mechanisms of cyclosporine A-induced apoptosis in rat hepatocyte primary cultures

In rat hepatocytes and isolated liver mitochondrial fractions, Cyclosporine A (CsA) is often used as a specific inhibitor of mitochondrial Ca(2+) release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 h. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 h with CsA at concentrations of 0, 10, 25, and 50 microM. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3, and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation and the number of TUNEL- and Annexin V-positive cells increased dose-dependently without any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 h of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEID-CHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events that trigger the apoptosis cascade. Transmission electron microscopy confirmed an increase in the number of necrotic cells after 20 h, but not after 4 h, compared with controls.     

Induction of metallothionein by steroids in rat primary hepatocyte cultures

The purpose of this study was to characterize the induction of metallothionein (MT) by steroids in rat primary hepatocyte cultures. Comparison of the characteristics of MT induction by a steroid (dexamethasone) to that by metals (Zn and Cd), examination of the involvement of the glucocorticoid receptor in the steroid induction of MT, and determination of the potency and effectiveness of a number of steroids were studied. In general, the patterns of MT induction by metals and steroids were quite different. For metals, the maximal MT induction (12- to 39-fold) was limited by toxicity whereas for steroids, a plateau in MT induction (fivefold) occurred at noncytotoxic concentrations. Steroids elicited an increase in MT at concentrations that were one-hundredth to one-thousandth less than that of metals. A combination of metal and steroid increased the induction of MT to a level higher than achieved by metal or steroid alone. The effectiveness of steroids at inducing MT was related to their ability to induce a specific glucocorticoid effect, induction of tyrosine aminotransferase. For specific classes of steroids, synthetic glucocorticoids were more potent than the metals in inducing MT, but endogenous glucocorticoids, mineralocorticoids, androgens, and estrogens were less potent than the metals. The concentration of corticosterone, the major endogenous glucocorticoid of rats, required to induce MT in hepatocytes was 100 times higher than concentrations achievable in the plasma of rats. In conclusion, in rat hepatocytes dexamethasone was a more potent but less effective inducer of MT than Zn or Cd; synthetic glucocorticoids were more potent but endogenous adrenalcorticoids (i.e., glucocorticoids, mineralocorticoids, androgens, and estrogens) were both less potent and less effective inducers of MT than were metals, suggesting that glucocorticoids may not be the mediator for stress-induced MT induction; and induction of MT by steroids correlated well with the induction of tyrosine aminotransferase, supporting the involvement of a hormone-receptor complex in the induction of MT by steroids.     

Biochemical assessment of the genotoxicity of the in vitro interaction between chlordecone and carbon tetrachloride in rat hepatocytes.

The genotoxic potential of the administration of carbon tetrachloride alone or carbon tetrachloride to chlordecone-pretreated rats was investigated using an in vivo-in vitro animal model and a battery of biochemical assays to measure DNA repair in rat hepatocytes. Whereas carbon tetrachloride alone was not genotoxic, chlordecone or chlordecone in combination with carbon tetrachloride was genotoxic. The need for further investigation into the mechanism underlying the interaction between chlordecone and carbon tetrachloride is indicated strongly by the results of the present study.     

In vivo protective effect of Porphyra yezoensis polysaccharide against carbon tetrachloride induced hepatotoxicity in mice

To study the protective effect and possible mechanism of Porphyra yezoensis polysaccharide (PYP) in hepatotoxicity mice, acute liver injury was successfully induced by injecting 0.2% carbon tetrachloride (CCl(4)) intraperitoneally. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and liver homogenate, content of malondialdehyde (MDA), activities of total superoxide dismutase (T-SOD) in liver were measured by biochemical methods. Liver index was calculated and pathological changes of the liver tissue were observed microscopically. PYP was found to significantly decrease the activities of ALT and AST (P<0.05), to remarkably lower the liver indexes and MDA level in hepatical tissues in mice (P<0.05), and to upregulated the lower T-SOD level in liver homogenate (P<0.01). Furthermore, histologic examination showed that PYP could attenuate and the extent of necrosis, reduce the immigration of inflammatory cells. PYP plays a protective action against hepatotoxicity induced by CCl(4) in mice, and its mechanisms may be related to free radical scavenging, increasing SOD activities and anti-lipid peroxide.     

In vitro toxicity assessment of cyclosporin A and its analogs in a primary rat hepatocyte culture model

The cytotoxicity of cyclosporin A (CsA), a widely used immunosuppressant drug, was evaluated in primary cultures of rat hepatocytes. Furthermore, the concentration-dependent (10(-7) to 10(-5) M) cytotoxic effects of the cyclosporin analogs, CsG, CsH, CsF, and of a major metabolite of CsA, CsA/M17, were assessed in an attempt to classify the different cyclosporin analogs according to their in vitro hepatotoxic potential. All compounds invariably inhibited the net release of taurocholate (de novo synthesized from cholate added to the extracellular medium). This sensitive functional marker did not discriminate between the structural analogs. In addition, all compounds inhibited, to various extents, the biosynthesis and secretion of proteins without affecting the uptake rate of the nonmetabolizable amino acid, alpha-aminoisobutyric acid. These functional changes occurred in the absence of overt irreversible cell damage (no leakage of lactic dehydrogenase up to 10(-5) M cyclosporin during 17 hr of incubation). The relative toxic potential of the drug congeners (CsG greater than CsA greater than CsH = CsF = CsA/M17) correlated well with the degree of their accumulation in the hepatocytes during exposure to equimolar drug concentrations.     

Microcystin-LR induced cellular effects in mammalian and fish primary hepatocyte cultures and cell lines: a comparative study

The impact of microcystin-LR, one of the most common cyanobacterial toxins, on liver and gut cells originating from mammals and fish was compared. Upon exposure of human and rainbow trout (Oncorhynchus mykiss) cell lines up to 2.5 microM microcystin-LR, no alteration in cell viability was observed as assessed with three fluorescent indicators dyes, CFDA-AM, Alamar Blue and neutral red. The lack of sensitivity of the trout cell lines coincided with an absence of detectable mRNA levels of organic anion transporter polypeptide (OATP), which is implicated in the uptake of microcystin-LR. In contrast to the cell lines, primary rainbow trout and mouse hepatocytes showed damage to subcellular structures, particularly the lysosomes, as indicated by neutral red. This led us to propose a thus far undetected role of lysosomes as targets and mediators of microcystin-LR elicited cellular damage. An inhibitor of OATP, rifampicin, partly protected hepatocytes from this damage. Yet, the sensitivity of rainbow trout hepatocytes rapidly declined in culture, accompanied by decreasing levels of OATP mRNA. The sensitivity of mouse hepatocytes toward microcystin-LR also declined in culture but overall was about 25-fold greater than that of the trout cells. These differences mirror those observed in vivo and suggest the use hepatocytes for deciphering the species differences.     

Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

Thyroxine (T₄)-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague–Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T₄-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T₄-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), β-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T₄-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T₄-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T₄-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T₄-UGT activities.     

In vitro and in vivo evaluation of the mechanisms of citalopram-induced hepatotoxicity

Even though citalopram is commonly used in psychiatry, there are several reports on its toxic effects. So, the current study was designed to elucidate the mechanisms of cytotoxic effects of in vitro and in vivo citalopram treatment on liver and the following cytolethal events. For in vitro experiments, freshly isolated rat hepatocytes were exposed to citalopram along with/without various agents. To do in vivo studies liver function enzyme assays and histological examination were performed. In the in vitro experiments, citalopram (500 µM) exposure demonstrated cell death, a marked elevation in ROS formation, mitochondrial potential collapse, lysosomal membrane leakiness, glutathione (GSH) depletion and lipid peroxidation. In vivo biochemistry panel assays for liver enzymes function (AST, ALT and GGTP) and histological examination confirmed citalopram (20 mg/kg)-induced damage. citalopram-induced oxidative stress cytotoxicity markers were significantly prevented by antioxidants, ROS scavengers, MPT pore sealing agents, endocytosis inhibitors, ATP generators and CYP inhibitors. Either enzyme induction or GSH depletion were concomitant with augmented citalopram-induced damage both in vivo and in vitro which were considerably ameliorated with antioxidants and CYP inhibitors. In conclusion, it is suggested that citalopram hepatotoxicity might be a result of oxidative hazard leading to mitochondrial/lysosomal toxic connection and disorders in biochemical markers which were supported by histomorphological studies.