Increased proteolysis after single-dose exposure with hepatotoxins in HepG2 cells.

Chronic ethanol consumption is associated with increased protein oxidation and decreased proteolysis in the liver. We tested the hypothesis that even single-dose treatment with ethanol or bromotrichloromethane causes increased protein oxidation and a distinct proteolytic response in cultured hepatocytes. HepG2 cells were treated for 30 min with ethanol, H(2)O(2) and bromotrichloromethane at various nontoxic concentrations. Protein degradation was measured in living cells using [35S]-methionine labeling. Protein oxidation, and 20S proteasome activity were measured in cell lysates. Oxidized proteins increased immediately after ethanol, H(2)O(2), and bromotrichloromethane exposure, but a further significant increase 24-h after exposure was observed only following ethanol and bromotrichloromethane treatment. All three reagents caused a significant increase of the overall intracellular proteolysis at rather low concentrations, which could be suppressed by the proteasome inhibitor lactacystin. A decline of proteolysis observed at higher-subtoxic-concentrations was not related to decreased proteasome activity. Preincubation with ketoconazole or 4-methylpyrazole completely prevented the ethanol- and bromotrichloromethane-induced but not the H(2)O(2)-induced protein oxidation and proteolysis, suggesting strongly an enzyme-mediated generation of reactive oxygen species. In conclusion single-dose exposure with ethanol or haloalkanes causes increased protein oxidation followed by an increased proteasome-dependent protein degradation in human liver cells.

Use of rat liver slices for the study of oxidative DNA damage in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells.

Tissue slices are a useful biological system for lipid peroxidation studies but their use for DNA damage studies is not well characterized. Hence, the present study investigates DNA damage in rat liver slices, in comparison with isolated rat liver nuclei and HepG2 human hepatoma cells, incubated with ferric nitrilotriacetate (Fe(III)-NTA), bromotrichloromethane (BrCCl(3)), bromobenzene (BrB) or 2-nitropropane (2-NP) at 37 degrees C for 2 hr. DNA damage was measured in slices, cells or nuclei after centrifugation as formation of as 8-hydroxy-2'-deoxyguanosine (8-OH-dGu) and loss of double-stranded (dsDNA) due to strand breakage using a fluorometric analysis of DNA unwinding (FADU). Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) released into the medium. The results show that in liver slices and isolated nuclei, Fe/NTA (1 mM/4 mM) induced high levels of TBARS but low levels of 8-OH-dGu, whereas the oxidant induced low levels of TBARS and no formation of 8-OH-dGu in HepG2 cells. In all three systems, inclusion of ascorbate caused dose-dependent formation of 8-OH-dGu, and the levels were similar between liver slices and HepG2 cells but were far higher in isolated nuclei. In liver slices the FADU assay was not applicable due to limited solubilization of DNA from the slice, whereas the assay detected significant loss of dsDNA in HepG2 cells and slight loss in isolated nuclei induced by Fe/NTA with or without ascorbate. Liver slices incubated with 1 mm BrCCl(3), BrB or 2-NP had elevated TBARS but had little or no formation of 8-OH-dGu; none of these oxidants induced lipid peroxidation or DNA damage in HepG2 cells. When liver slices obtained from rats injected with diethylmaleate (to deplete GSH) were incubated with BrCCl(3), BrB or 2-NP, levels of TBARS and 8-OH-dGu increased markedly. Similarly, HepG2 cells with decreased GSH showed marked elevation of TBARS and loss of dsDNA induced by these oxidants, although no formation of 8-OH-dGu was detected. The present study demonstrates the usefulness and limitations of liver slices for DNA damage studies and the importance of cellular GSH in the protection of DNA against environmental toxicants.

Suicidal inactivation of haemoproteins by reductive metabolites of halomethanes: a structure-activity relationship study.

Human haemoglobin (Hb), methaemalbumin (MHA) or rat liver microsomal cytochrome P-450 (P-450) were incubated anaerobically at microM concentrations with 1 mM carbon tetrachloride (CCl4), trichlorobromomethane (CCl3Br), chloroform (CHCl3) or methylene chloride (CH2Cl2) in presence of 1 mM sodium dithionite as the reducing agent. At the end of a 5-min incubation, haem was measured by various methods, i.e. binding spectrum with CO, pyridine-haemochromogen haem assay and porphyrin fluorescence, and compared for the four analogues. Statistically significant losses were observed, with all three haemo-protein systems, for CCi3Br, CCl4 and CHCl3, but not CH2Cl2. For Hb, the loss was greater with CCl3Br (haem assay, 63%; porphyrin fluorescence, 48%; CO binding, 24%) than with CCl4 (haem assay, 31%) or CHCl3 (haem assay, 13%). On the other hand, with MHA, CCl4 gave a dramatic loss (haem assay, 88%; porphyrin fluorescence, 83%; CO binding, 67%), which was greater than that observed with CCl3Br (haem assay, 49%; porphyrin fluorescence, 38%; CO binding, 25%). No loss was found with CHCl3. Finally, with microsomes, the inactivation was larger with CCl4 (CO binding, 58%; haem assay, 50%; porphyrin fluorescence, 33%) than with CCl3Br (CO binding, 33%; haem assay, 10%) or CHCl3 (haem assay, 9%; CO binding, 6%). In a separate set of similar experiments, an ion-pairing reverse phase HPLC method showed the formation of substrate-dependent hae-derived products during incubation of CCl3Br with Hb or microsomes, and of CCl4 with Hb. A correlation between potential for free radical formation (CCl3Br > CCl4 > CHCl3 > CH2Cl2) and extent of haem inactivation was observed with all methods for Hb, but not for microsomal P-450 or MHA. The results indicate that these halomethanes may be activated differently by different haemoproteins and suggest that their potential ability to undergo reductive metabolism may not be the only critical factor involved in P-450 haem inactivation by these chemicals.

Potentiation of oxidative damage to rat red blood cells by the concurrent presence of t-butyl hydroperoxide and bromotrichloromethane.

Recently potentiation of oxidative damage in rat red blood cells (rRBC) incubated with t-butylhydroperoxide (BHP) in combination with bromotrichloromethane (BrCCI3) was demonstrated. The mechanism by which this combination (BrCCI3/BHP) potentiates the oxidative damage to rRBC was investigated in this study. When rRBC were incubated with 0.1 mM BHP, 0.5 mM BrCCI3, or the two combined, BrCCI3/BHP-potentiated lipid peroxidation and hemolysis were further enhanced under anaerobic conditions. However, the potentiation of lipid peroxidation was abolished by heating or trypsin digestion of rRBC. Electron spin resonance (ESR) studies demonstrated an increase of alkoyl radical induced by BrCCI3/BHP in rRBC, and this increase was abolished by heating or predigestion of hemolysates with trypsin. The inhibition of lipid peroxidation by diphenylamine (which reacts with alkoxyl radicals but not peroxyl radicals) suggests an important role of alkoxyl radicals. Overall, the present findings demonstrate that the increase in radical-related oxidative damage, possibly mediated by proteinlike materials, may be at least partially responsible for the potentiation of damage to rRBC induced by BrCCI3/BHP, and perhaps by BrCCI3. Although the in vivo significance of these results remains to be investigated, it seems likely that halocarbon toxicity may be amplified by elevated levels of lipid peroxide in blood.

The role of glutathione and protein thiols in CBrCl3-induced cytotoxicity in isolated rat hepatocytes.

The role of glutathione (GSH) and protein thiols in the pathobiochemical process of CBrCl3 cytotoxicity was investigated in isolated hepatocytes. Administration of 0.5, 1.0 and 1.5 mmol/l CBrCl3 affected cellular viability as assessed by trypan blue exclusion, release of lactate dehydrogenase and loss of intracellular potassium in a dose-dependent manner. Intracellular glutathione and the capacity to reduce 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazolium bromide (MTT, thiazolyl blue) decreased almost independently of the CBrCl3 concentration. Protein thiols were not markedly oxidized in the presence of CBrCl3. However, compromising cellular defence mechanisms by either inhibition of glutathione regeneration or depletion of glutathione enhanced the cytotoxicity of CBrCl3 and induced a loss of protein thiols in the late phase of cellular injury. Under these conditions the thiol-dependent Na+,K+ATPase revealed high sensitivity towards CBrCl3. Thus, glutathione proved to exert effective cytoprotection, and sulfhydryl groups of particular proteins were supposed to be an important target of radical attack.

The interactive toxicity of CHCl3 and BrCCl3 in precision-cut rat liver slices.

The interactive toxicity of two nontoxic concentrations of chloroform (CHCl3) and bromotrichloromethane (BrCCl3) was examined in precision-cut rat liver slices. Liver slices were prepared from male Sprague-Dawley rats (220-250 g) pretreated with phenobarbital for 4 days. Toxicants were administered 1 hr apart. Intracellular K+ levels were similar to untreated controls in slices treated with 0.2 mM CHCl3 or 0.125 microliters (0.25 mg, 1.26 mumol) BrCCl3 alone, indicating that these concentrations were nontoxic. However, addition of both toxicants, irrespective of order, resulted in a time-dependent loss of intracellular K+ which was significant at 9 hr following administration. This was interpreted as evidence of synergistic toxicity. Cytochrome P450 loss was significant as early as 3 hr following exposure to BrCCl3, alone or when added with CHCl3. This loss may be attributed to BrCCl3-induced suicide inactivation of cytochrome P450. Centrilobular hepatocytes may be more susceptible to the interactive toxicity of CHCl3 and BrCCl3. Activity of enzymes found predominantly in this area was significantly decreased in slices exposed to both toxicants relative to controls. Conversely, activity of enzymes found predominantly in the periportal region was similar to that of untreated and treated controls. Interactive toxicity of BrCCl3 and CHCl3 was not a consequence of increased lipid peroxidation or depletion of slice glutathione content. Further studies need to be conducted to elucidate the mechanisms mediating the interactive toxicity of BrCCl3 and CHCl3.

Trichlorobromomethane-induced changes of purine nucleotides in hepatocytes.

The changes in nucleotide content during CBrCl3 treatment were investigated. In the first 5-10 minutes a significant ATP decrease was detected. The GTP loss leading to 57% of the initial level after 10 min surpasses the ATP loss which leads to 70% of the initial value after 10 min. The increase in uric acid is not only the result of CBrCl3 induced reaction, because of no significant changes in adenine and hypoxanthine values. The uric acid pool reflected different influx and efflux processes. These changes were compared with nucleotide degradation and accumulation of nucleotide degradation products during anoxia.

Determination of 4-hydroxynonenal by high-performance liquid chromatography with electrochemical detection.

4-Hydroxy-trans-2-nonenal (HNE) is a highly reactive product of lipid peroxidation originating from the break-down of phospholipid-bound polyunsaturated fatty acids of cellular membranes. Despite its biological relevance, this aldehyde is only occasionally determined due to the complexity of previously described procedures. Here we present a simple and very sensitive method for the detection of HNE in biological samples. The method is based on the measurement of the 2,4-dinitrophenylhydrazone (DNPH) of the aldehyde by electrochemical detection after separation by reverse-phase high-performance liquid chromatography (HPLC). The greater sensitivity of this procedure as compared to the ultraviolet detection method commonly employed to measure DNPH derivatives of aldehydes after HPLC will allow the detection of HNE below the pmol level. The detection of HNE is highly reproducible even in normal tissues and cells. Increased amounts of HNE were detected in the livers of animals intoxicated with prooxidant agents such as carbon tetrachloride, bromotrichloromethane or bromobenzene. An exponential increase in HNE (and in malondialdehyde) was measured in peroxidizing liver microsomes (in the NADPH/Fe-dependent system). The method is also suitable for the study of very small samples, since HNE could be detected in approximately 1 million cultured cells (polyoma virus-transformed baby hamster kidney fibroblasts); the level rose after exposure of the cells to a Fe3+/ADP prooxidant system.

In vivo studies on halogen compound interactions. IV. Interaction among different halogen derivatives with and without synergistic action on liver toxicity.

The liver toxicity of several halogen compound mixtures have been tested. The compounds were selected on the basis of their metabolic pathways: carbon tetrachloride (CT) and trichlorobromomethane (TCBM) undergo a dehalogenation via P450-dependent enzyme system, 1,2-dichloroethane (DCE) and 1,2-dibromoethane (DBE) are mainly conjugated with the cytosolic glutathione (GSH) by means of the GSH-S-transferase. The mixture TCBM+DBE shows a more than additive action on lipid peroxidation and liver necrosis. TCBM, like CT, reduces the hepatic level of GSH-S-transferase, increasing the amount of DBE available for cytochrome P450-dependent metabolism, with the production of toxic metabolites. Thus, the behavior of the mixture TCBM+DBE is very similar to that of the mixture CT+DBE, previously reported. Mixtures composed of CT+TCBM and DCE+DBE do not show any synergistic effect on liver toxicity. The results allow one to conclude that the toxicity of mixtures of halogen compounds can be partly predicted on the basis of their metabolic pathways. When the metabolism is quite different, a synergistic toxicity can occur if one pathway interferes with a detoxification mechanism of the other compound. If the two metabolisms are very similar they produce, at most, an additive toxicity.

Lipid peroxidation and irreversible damage in the rat hepatocyte model. Protection by the silybin-phospholipid complex IdB 1016.

IdB 1016 is a new silybin-phospholipid complex which is more bioavailable than the flavonoid silybin itself and displays free radical scavenging and antioxidant properties in liver microsomes. We report here that the addition of increasing concentrations of IdB 1016 to isolated rat hepatocytes caused a dose-dependent inhibition of lipid peroxidation induced by ADP-Fe3+ or cumene hydroperoxide. Moreover, IdB 1016 at the concentration which completely prevented MDA formation also protected isolated hepatocytes against the toxicity of pro-oxidant agents such as allyl alcohol, cumene hydroperoxide and bromotrichloromethane, without interfering with the activation mechanism of these xenobiotics. Similar protection was also obtained in hepatocytes prepared from animals pretreated in vivo with IdB 1016 while rat supplementation with pure silybin was totally inefficient. These results indicate IdB 1016 as being a potentially useful protective agent against free radical-mediated toxic liver injury.

Phospholipase A2 activation and cell injury in isolated rat hepatocytes exposed to bromotrichloromethane, chloroform, and 1,1-dichloroethylene as compared to effects of carbon tetrachloride.

It was found that the four toxigenic agents, CCl4, CHCl3, CBrCl3, and 1,1-dichloroethylene (vinylidene chloride) all share the property of activating phospholipase A2 (PLA2) of isolated hepatocytes in suspension, as determined over a 60- or 120-min time period. In all cases, PLA2 activation, measured as the appearance of lysophosphatidylethanolamine, preceded the release of lactic dehydrogenase during incubation of the cells at 37 degrees C. It is concluded that for these halogenated hydrocarbons phospholipase A2 activation may be part of the chain of causality leading from initial bioactivation to ultimate cell death.

Brazilin protects cultured rat hepatocytes from BrCCl3-induced toxicity.

Brazilin, the main constituent of Caesalpinia sappan, is an antioxidative substance that has catechol moiety in its chemical structure. Considering the antioxidant-activity of brazilin, it was expected to have protective effects on the toxicities of radical generating chemicals. The incubation of rat hepatocytes with BrCCl3 resulted in significant increase in lipid peroxidation, leakage of cytoplasmic enzymes and cytoplasmic glutathione depletion. The BrCCl3-induced toxicities on hepatocytes were reduced by the treatment of brazilin. Brazilin has been also proved to have a protective effect on the BrCCl3-induced depression of microsomal calcium sequestration activity. These results indicate that brazilin plays a protective role in BrCCl3-induced hepatocyte injury of the rat.

Potentiation of BrCCl3 hepatotoxicity by chlordecone: biochemical and ultrastructural study.

Previous work has established that chlordecone (CD) potentiates the hepatotoxicity of BrCCl3. This interaction occurs at nontoxic levels of CD and BrCCl3. The present research was designed to investigate the mechanism governing the pathogenesis of potentiated hepatic injury and lethality induced by a low dose of BrCCl3 after dietary pretreatment with 10 ppm of CD for 15 days. On Day 16, a single dose of BrCCl3 (30 microliters/kg) was administered ip to rats maintained either on normal diet (ND) or on a diet contaminated with 10 ppm CD. Blood and liver samples were collected at 0, 3, 6, 12, 24, 36, and 48 hr after the halomethane administration for biochemical (ATP, bilirubin, glycogen) and for ultrastructural studies. A continuous increase in serum bilirubin and decrease in hepatic ATP and glycogen were observed in CD + BrCCl3 combination, indicating progressive injury, but not in other treatment groups. In ND + BrCCl3 combination, all biochemical indices were either normal or close to normal after 36 hr, suggesting complete recovery from hepatotoxicity. The most extensive ultrastructural changes characteristic of halomethane hepatotoxicity (necrosis, ballooned cells, and dilation of rough endoplasmic reticulum) were observed after the CD + BrCCl3 combination treatment. The progressive and early depletion of hepatic ATP and glycogen, and the progressive increase in toxicity along with decreased cell division in CD + BrCCl3-treated rats, indicate the association of compromised energy status and suppression of cell division and tissue repair in CD-potentiated BrCCl3 toxicity. These findings suggest that the suppression of stimulated hepatocellular regeneration results in the loss of the essential mechanism of tissue repair leading to continuation of the toxic liver injury associated with the CD + BrCCl3 combination treatment.

PB-PK derived metabolic constants, hepatotoxicity, and lethality of BrCCl3 in rats pretreated with chlordecone, phenobarbital, or mirex.

Pharmacokinetic modeling has been very useful in examining the complex relationships between exposure concentration and target tissue dose. This study utilizes a physiologically based pharmacokinetic (PB-PK) modeling approach for assessing the metabolism of BrCCl3 and to investigate its relationship with hepatotoxicity and lethality. Male Sprague-Dawley rats maintained for 15 days on normal diet (control), or on diets containing either chlordecone (CD, 10 ppm), phenobarbital (PB, 225 ppm) or mirex (M, 10 ppm), were used in gas uptake studies to determine the kinetic constants of BrCCl3 metabolism. Four initial concentrations of BrCCl3 at approximately 30, 200, 700, and 3000 ppm were used for each group. The uptake data were analyzed by computer simulation using a PB-PK model containing relevant tissue solubilities and physiological parameters as well as an equation describing the behavior of BrCCl3 in the closed chamber atmosphere. Liver injury was assessed by serum enzyme elevations (alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase) and histopathological examination, at 24 hr after the exposure to BrCCl3. Another group of similarly pretreated rats was exposed to BrCCl3 and observed over a 14-day period for mortality. Dietary exposures resulted in increased Vmaxc value for BrCCl3 metabolism as compared to control (3.55 +/- 0.14 mg/hr/kg) for PB (8.52 +/- 0.28 mg/hr/kg) and M (5.06 +/- 0.19 mg/hr/kg) but not for CD (3.92 +/- 0.19 mg/hr/kg). Kfc, the first-order rate constant for BrCCl3 metabolism, was decreased after PB (12.9 +/- 0.5 hr-1/kg) and increased after M (17.6 +/- 0.5 hr-1/kg), but unchanged after CD (15.5 +/- 0.6 hr-1/kg) exposure as compared to control (15.0 +/- 0.3 hr-1/kg). The total amount of BrCCl3 metabolism at any initial concentration employed remained unchanged in all the pretreated groups as compared to control. However, the amount of BrCCl3 metabolized through saturable pathway only, at higher initial concentrations, was increased in the PB and M pretreated groups, but not in the CD pretreated group. It is concluded that the rates of metabolism of BrCCl3 were unchanged after CD pretreatment as compared to control, while PB and M pretreatment alter both the saturable and first-order rates. Serum enzymes were significantly increased in all the groups after exposure to BrCCl3 at 200 and 700 ppm concentrations. The increase was more pronounced in PB and M pretreated groups as compared to control and CD pretreated groups. Similarly, histopathological examination of liver showed alterations in the lobular architecture, the extent of alterations being dependent on the dose of BrCCl3 and the pretreatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Histological evidence for dissociation of lipid peroxidation and cell necrosis in bromotrichloromethane hepatotoxicity in the perfused rat liver.

Bromotrichloromethane (CBrCl3)-induced hepatic lipid peroxidation and cell necrosis were studied histologically and biochemically, using isolated perfused livers from phenobarbital-pretreated rats. Lipid peroxidation was assessed by fuchsin staining of the liver slices and release of thiobarbituric acid reactive substances (TBARS) into the perfusate; necrosis was assessed by trypan blue uptake and lactate dehydrogenase (LDH) leakage. A good correlation was observed between the Schiff-positive reaction and TBARS release under various experimental conditions, supporting the validity of the fuchsin staining method for histological detection of lipid peroxidation. Lobular localization of lipid peroxidation and necrosis was as follows: Under high oxygen supply (95% O2-saturated buffer), infusion of CBrCl3 caused the Schiff-positive reaction in the pericentral to midzonal hepatocytes, irrespective of the direction of perfusion, but did not produce necrosis. Under low oxygen supply (20% O2) with retrograde perfusion, dissociation of lipid peroxidation and necrosis was observed, i.e., trypan blue uptake in the periportal zones and Schiff-positive staining in the pericentral hepatocytes. Thus, lipid peroxidation by itself may have a relatively minor role in the development of CBrCl3-induced acute hepatic cell death.

Hepatotoxicity and lethality of halomethanes in Mongolian gerbils pretreated with chlordecone, phenobarbital or mirex.

The hepatotoxic and lethal effects of CBrCl3, CCl4 and CHCl3 were investigated in gerbils with or without prior exposure to dietary chlordecone (CD), phenobarbital (PB) and mirex (MX) at 10, 225 and 10 ppm, respectively, for 15 days. Gerbils were quite sensitive to these halomethanes (48 h LD50: 20, 80 and 400 microliters/kg, respectively). CD, known to potentiate hepatotoxic and lethal effects of halomethanes in rats, failed to potentiate the toxic effects of any of these three halomethanes in gerbils. PB and MX were also ineffective. Since stimulation of early hepatocellular regeneration has been shown to be responsible for the recovery from the toxicity of a low dose of CCl4, liver cell regeneration and tissue repair were studied in gerbils after CCl4 administration. The objectives of these studies were to investigate the possible reasons for the high sensitivity of gerbils to halomethane toxicity and to investigate the mechanism for their refractoriness to CD-potentiated halomethane toxicity. A low and a high dose of CCl4 (15 and 80 microliters/kg, i.p. respectively) were used to study the time-course of liver injury in gerbils pretreated with or without CD. The low dose of CCl4 stimulated cellular regeneration as indicated by the increase of 3H-thymidine (3H-T) incorporation in hepatic nuclear DNA. The cellular regeneration and tissue repair activities resulted in complete recovery from the limited liver injury in both CD-pretreated and control gerbils. In contrast to rats, however, the process of cell division in gerbils occurred much later, 2 days after CCl4 administration. Evidence from histomorphometric studies was consistent with serum enzyme and 3H-T incorporation data.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanisms for bromotrichloromethane cytotoxicity in isolated rat hepatocytes.

1. Bromotrichloromethane added to isolated rat hepatocytes resulted in increased cell death as determined by trypan blue uptake. Toxicity increased in a concentration-dependent fashion between 2.0-5.0 M bromotrichloromethane. 2. Lipid peroxidation (malondialdehyde) increased in a time-dependent fashion but in contrast to toxicity reached a maximum level at 2.0 mM bromotrichloromethane. 3. Hypoxia increased the toxicity of bromotrichloromethane three-fold but only decreased the amount of lipid peroxidation to a small degree. 4. In spite of this poor correlation between toxicity and lipid peroxidation, the antioxidant butylated hydroxyanisole and the iron chelator desferal protected the cells from toxicity under both aerobic and hypoxic conditions and prevented lipid peroxidation. 5. During treatment with bromotrichloromethane, cellular glutathione levels slowly decreased and oxidized glutathione appeared in the media. The addition of cystine to the incubation media prevented the formation of extracellular oxidized glutathione, indicating that cellular glutathione had leaked from the cell during treatment and was oxidized in the incubation media. Although this suggested that glutathione does not play a protective role against bromotrichloromethane toxicity, diethyl maleate-pretreatment of the cells to decrease glutathione levels markedly increased bromotrichloromethane toxicity. 6. The addition of ascorbic acid to the incubation media increased bromotrichloromethane toxicity. This was attributed to the reductive activation of bromotrichloromethane in an iron and oxygen-dependent reaction. 7. It was concluded that peroxidation of essential phospholipids contributes to bromotrichloromethane-induced hepatocyte cytotoxicity.

Bromotrichloromethane hepatotoxicity. The role of stimulated hepatocellular regeneration in recovery: biochemical and histopathological studies in control and chlordecone pretreated male rats.

It has been shown that BrCCl3 is a more potent hepatotoxin than CCl4. Pretreatment with nontoxic dietary levels of chlordecone (CD) results in amplification of BrCCl3 hepatotoxicity. The objective of this research was to investigate and compare the histopathological alterations during a time course after a low dose of BrCCl3 alone and in combination with dietary CD. Male Sprague-Dawley rats were maintained on 10 ppm dietary CD or normal diet for 15 days. On day 16, they received a single ip dose (30 microliters/kg) of BrCCl3 in corn oil (CO) vehicle or corn oil alone. Blood and liver samples were collected at 0, 3, 6, 12, 24, 36, 48, 72, 96, and 120 hr for serum enzymes and histopathological examination, respectively. Serum enzymes (SDH, ALT, AST) were significantly (p less than 0.05) elevated in rats receiving the CD + BrCCl3 combination in comparison to BrCCl3 alone. For 48 hr, a continuous increase in serum enzyme activities was detected in rats treated with CD + BrCCl3 combination, but not in the rats receiving other treatments (ND + BrCCl3, ND + CO, or CD + CO). The most extensive hepatolobular necrosis was observed in rats treated with the CD + BrCCl3 combination. Thirty-six hr after the administration of BrCCl3 to rats maintained on normal diet, high mitotic activity was observed, which continued through 72 hr resulting in complete restoration of hepatolobular structure. In contrast, rats receiving the combination of CD + BrCCl3 exhibited minimal and belated hepatomitotic activity for a short period of time, resulting in progressive hepatic failure, culminating in animal death. In conclusion, hepatotoxicity of a low dose of BrCCl3 alone appeared to be overcome via stimulated hepatocellular regeneration and hepatolobular restoration. CD appears to amplify BrCCl3 hepatotoxicity via interference with this hormetic mechanism, permitting a progressive and continued hepatic injury leading to complete hepatic failure, culminating in animal death.

Perturbations in polyamines and related enzymes following chlordecone-potentiated bromotrichloromethane hepatotoxicity.

The mechanism by which chlordecone (CD) amplifies the hepatotoxicity of halomethanes such as CCl4, CHCl3, and BrCCl3 has been a subject of intense study. Recent work has shown that suppression of hepatocellular regeneration leads to accelerated progression of liver injury leading to complete hepatic failure due to an unusual interaction between individually nontoxic low-dose combination of CD and CCl4. Since polyamines are involved in cell division, their levels reflect the extent to which there is suppression of hepatocellular regeneration during CD and CCl4 interaction. The present studies were designed to investigate the polyamine levels and associated enzymes in livers of rats treated with BrCCl3 alone or CD and BrCCl3 low-dose combination in order to confirm whether the sequence of events of hepatotoxicity is similar to that seen in CCl4 toxicity or that seen during CD and CCl4 interaction. The extent of liver toxicity in rats fed 10 ppm chlordecone (CD) for 15 days prior to the injection of a single low dose of BrCCl3 (15 microL/kg body weight) or after exposure to a high dose of BrCCl3 (80 microL/kg body weight) without CD pretreatment, was similar 6 and 24 hr later as assessed by plasma transaminase levels. There was also an increase in transaminase levels, in rats exposed to a single low dose of BrCCl3 alone (15 microL/kg body weight) but this increase was far below the high-dose exposure alone or the combination treatment. Hepatic levels of ornithine decarboxylase, S-adenosylmethionine decarboxylase, N1-acetylputrescine, N1-acetylspermidine, putrescine, spermidine, and spermine at the end of 24 hr increased after exposure to a low dose of BrCCl3 alone as compared to exposure to a high dose alone or the low-dose combination of CD and BrCCl3. Liver spermidine N1-acetyltransferase was elevated at 2, 6, and 24 hr after exposure to a high dose of BrCCl3 alone as compared to treatment with a low-dose combination of CD and BrCCl3 suggesting decreased synthesis of this enzyme, in spite of a greater need as seen from liver transaminase levels. In general, it was observed that there is significant elevation in some polyamines and related enzymes during toxicity of a low dose of BrCCl3 which seemed to stabilize within 24 hr. This was not observed with the other two groups of rats exposed either to BrCCl3 high dose alone or the low-dose combination of CD and BrCCl3.(ABSTRACT TRUNCATED AT 400 WORDS)