Acute hepatotoxicity and lethality of CCl4 in chlordecone-pretreated rats

In a subchronic dietary pretreatment protocol chlordecone (CD) is a powerful potentiator of CCl4 hepatotoxicity, as indicated by biochemical, hepatofunctional, histopathological, and lethality parameters. The purpose of this investigation is to further explore the CD + CCl4 interaction in an acute CD pretreatment protocol and to compare the two pretreatment protocols in terms of their effect upon quantitative histopathology, serum enzymes, and lethality. Groups of four male rats received one of the following four pretreatments: chlordecone (10 mg/kg; single po), mirex (10 mg/kg; single po), phenobarbital (PB) (80 mg/kg/day for 2 successive days; ip in 0.9% saline), or corn oil vehicle (1 ml/kg; single po). Twenty-four hours later, the rats were given a single ip injection of CCl4 (0.1 ml/kg). Twenty-four hours after CCl4 administration, serum enzymes (SGPT, SGOT, and ICD) were measured and the livers removed and fixed in 10% buffered formalin for histological evaluation. The LD50 were determined by the method of moving averages. CD + CCl4 was the most hepatotoxic combination, in terms of serum enzyme elevations and lethality followed by PB + CCl4. The PB + CCl4 combination caused a greater degree of hepatocyte necrosis. These findings indicate that the acute pretreatment with CD enhances hepatotoxicity and the lethality of CCl4 in a fashion qualitatively similar to the subchronic pretreatment protocol.     

Chlordecone-induced potentiation of carbon tetrachloride hepatotoxicity: a morphometric and biochemical study

The present study, conducted over a time course of 36 hr after CCl4 administration, describes sequential morphometric and biochemical changes which occur in livers of rats exposed to a combination of low levels of chlordecone (10 ppm for 15 days) and a single ip injection of CCl4 (0.1 ml/kg). Those changes were compared to hepatic alterations which occur in rats that received the same dose of chlordecone or CCl4 alone. Biochemical studies showed only trivial increases in levels of glutamic-pyruvic transaminase (GPT), glutamic-oxalacetic transaminase (GOT), and moderate but temporary increases in isocitrate dehydrogenase (ICD) after CCl4 alone. The combination of chlordecone and CCl4 resulted in significantly greater elevations of all three serum enzymes at all time intervals examined. Morphometric data showed no difference between normal diet controls and animals exposed to chlordecone alone as far as numerical density of hepatocytes or volume densities of hepatocytes with glycogen, lipid, dilated rough endoplasmic reticulum (RER), pyknosis, or mitoses. Morphometric analysis of livers from animals that received CCl4 alone showed decreases in numerical density, temporary decrease in percentage of hepatocytes containing glycogen, an increase in hepatocytes containing lipid, temporary increase in hepatocytes with dilated RER, and temporary increases in pyknotic nuclei. Soon after the initial hepatic injury was histologically evident between 4 and 6 hr, the number of mitoses increased dramatically and this progressed until complete recovery from CCl4 damage. From all indices of damage, complete recovery was evident by 36 hr after CCl4 administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chlordecone-induced potentiation of carbon tetrachloride hepatotoxicity: a light and electron microscopic study

Previous studies have shown that a chlorinated pesticide, chlordecone (Kepone), greatly potentiates carbon tetrachloride (CCl4) hepatotoxicity and lethality (Curtis, L.R., Williams, W.L., and Mehendale, H.M. (1979). Toxicol. Appl. Pharmacol. 51, 283-293; Curtis, L.R., and Mehendale, H.M. (1980). Drug Metab. Dispos. 8, 23-27). The present study describes sequential morphologic changes which occurred in livers of rats given a "nontoxic" level of chlordecone (10 ppm for 15 days) followed by a single injection of CCl4 (0.1 ml/kg). The hepatic alterations were examined 1 to 36 hr after exposure of the rats to CCl4. Those changes were compared to hepatic alterations which occurred in rats that received the same dose of chlordecone (10 ppm for 15 days) or a single injection of CClr (0.1 ml/kg) alone. The only change noted in livers from rats that received chlordecone alone was focal increase in smooth endoplasmic reticulum (SER) of hepatocytes at 24 hr and continuing throughout the time course of the experiment. Livers from animals that received CCl4 alone showed morphologic changes at 6 hr consisting of glycogen loss, increase in SER, and dilatation of rough endoplasmic reticulum (RER) in pericentral hepatocytes. Accumulation of small lipid droplets was also noted in midzonal hepatocytes. After 6 hr, there was no further increase in severity of injury. At 12 hr recovery was noticeable and, by 36 hr, livers from the CCl4 group appeared normal. Prior administration of chlordecone greatly potentiated pathologic changes in livers of animals that received CCl4. By 4 hr, there was total loss of glycogen in hepatocytes throughout the entire lobule. Small lipid droplets were present in pericentral, midzonal and periportal hepatocytes. Hepatocytes with extremely dilated RER were randomly scattered throughout the entire lobule. At 6 hr, there was further accumulation of lipid in the form of large droplets in hepatocytes. Focal, necrotic cells surrounded by polymorphonuclear leukocytes were randomly distributed throughout the lobule. The number of necrotic foci had progressively increased at the 12- and 24-hr intervals. By 36 hr, confluent areas of necrosis in pericentral and midzonal areas were observed in livers of some animals. This study indicates that although the combination of chlordecone and CCl4 produces much greater hepatic injury resembling damage due to a massive dose of CCl4, histologically, some differences in the progression and distribution of hepatocellular damage within the lobular architecture of the liver are evident.     

Potentiation of halomethane hepatotoxicity by chlordecone: A hypothesis for the mechanism

A major toxicological issue today is the possibility of unusual toxicity due to interaction of toxic chemicals upon environmental or occupational exposures to two or more chemicals, at ordinarily harmless levels individually. While some laboratory models exist for such interactions for the simplest case of only two chemicals, progress in this area has suffered for want of a model where the two interactants are individually nontoxic. One such model is available, where prior exposure to nontoxic levels of the pesticide Kepone (chlordecone) results in a 67-fold amplication of CCl4 lethality in rats. Extensive hepatotoxicity observed in this interaction is characterized by histopathological alterations, perturbation of related biochemical parameters and is followed by complete hepatic failure. This propensity for chlordecone to potentiate hepatotoxicity of halomethanes such as CCl4, CHCl3, and BrCCl3 has been a subject of intense study to unravel the underlying mechanism. Mechanisms such as induction of microsomal cytochrome P-450 by chlordecone and greater lipid peroxidation are inadequate to explain the remarkably powerful potentiation of halomethane toxicity. Compelling experimental evidence supports the hypothesis that hepatocellular division during early time points after the administration of CCl4 is an important determinant of the progression (or repair of it) of the liver injury and consequent destruction (or restoration) of the hepatolobular architecture and function. This paper advances a hypothesis for the mechanism of hepatotoxic and lethal effect of CCl4 as being primarily related to the accelerated progression of liver injury due to suppressed hepatocellular regeneration and hepatolobular restoration. This is in contrast to the widely accepted putative mechanism, one which invokes only bioactivation followed by runaway lipid peroxidation as the events determining the course of the progressive phase of liver injury. The concept being advanced in this paper accepts bioactivation (and perhaps lipid peroxidation) as the primary initiating events of cell injury, but maintains that they are not the determinants of the progressive phase of liver injury. The biological issue of whether the cells are incapacitated from regenerating is the determinant of the progression of liver injury, and therefore, the ultimate outcome of hepatotoxicity and lethality.     

Pivotal role of hepatocellular regeneration in the ultimate hepatotoxicity of CCl4 in chlordecone-, mirex-, or phenobarbital-pretreated rats.

Our earlier histomorphometric and biochemical studies suggested that the progressive phase of the interactive toxicity of chlordecone (CD) + CCl4 involves suppression of hepatocellular regeneration. The objective of the present work was to correlate hepatocellular regeneration with CCl4 (100 microliters/kg)-induced hepatotoxicity in rats maintained for 15 days on a normal (N) diet, relative to the regenerative response in rats maintained on a diet containing either 10 ppm CD, 225 ppm phenobarbital (PB), or 10 ppm mirex (M). Hepatocellular regeneration was assessed by measuring DNA and 3H-thymidine (3H-T) incorporation, followed by autoradiographic analysis of liver sections. Hepatotoxicity was assessed by measuring plasma transaminases (aspartate and alanine) followed by histopathological observations of liver sections for necrotic, swollen, and lipid-laden cells. Lethality studies were also carried out to assess the consequence of hepatotoxicity on animal survival. Dietary 10 ppm CD potentiated the hepatotoxicity of CCl4 to a greater extent than PB or M, as evidenced by elevations in plasma enzymes. Although the serum enzymes were significantly elevated in PB rats in contrast to the slight elevations in N and M rats, they returned to normal levels by 96 hr. However, serum enzyme elevations in CD rats were progressive with time until death of the animals. Actual liver injury by CCl4 was greater in PB- than in CD-pretreated rats, as evidenced by histopathological observations. A 100% mortality occurred in CD-pretreated rats at 60 hr after CCl4 administration, whereas no mortality occurred in either N-, M-, or PB-pretreated rats, indicating recovery from liver injury. Hepatocellular nuclear DNA levels were significantly decreased starting at 6 hr after CCl4 administration to CD-pretreated rats, but not in M- or PB-pretreated rats. 3H-T incorporation into nuclear DNA as well as percentage of labeled cells showed a biphasic increase in N rats: 1 at 1-2 hr, and the other at 36-48 hr after CCl4 administration. However, only 1 peak of 3H-T incorporation at 36-48 hr was observed in the CD + CCl4 combination, which was also significantly lower when compared to that observed after the M or PB + CCl4 combination treatments. These findings suggest that there is recovery in N-, PB-, or M-pretreated rats from CCl4-induced injury by virtue of the stimulated hepatocellular regeneration and tissue repair.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tolerance of aged Fischer 344 rats against chlordecone-amplified carbon tetrachloride toxicity

We have investigated the effects of chlordecone 1(CD)+CCl4 combination in adult (3 months), middle aged (14 months), and old aged (24 months) male Fischer 344 (F344) rats. After a non-toxic dietary regimen of CD (10 ppm) or normal powdered diet for 15 days, rats received a single non-toxic dose of CCl4 (100 microl/kg, i.p., 1:4 in corn oil) or corn oil (500 microl/kg, i.p.) alone on day 16. Liver injury was assessed by plasma ALT, AST, and histopathology during a time course of 0-96 h. Liver tissue repair was measured by [3H-CH3]-thymidine (3H-T) incorporation into hepatic nuclear DNA and proliferating cell nuclear antigen (PCNA) immunohistochemistry. Hepatomicrosomal CYP2E1 protein, enzyme activity, and covalent binding of 14CCl4-derived radiolabel were measured in normal and CD fed rats. Exposure to CCl4 alone caused modest liver injury only in 14- and 24-month-old rats but neither progression of injury nor mortality. The CD+CCl4 combination led to 100% mortality in 3-month-old rats by 72 h, whereas none of the 14- and 24-month-old rats died. Both 3- and 14-month-old rats exposed to CD+Cl4 had identical liver injury up to 36 h indicating that bioactivation-mediated CCl4 injury was the same in the two age groups. Thereafter, liver injury escalated only in 3-month-old while it declined in 14-month-old rats. In 24-month-old rats initial liver injury at 6 h was similar to the 3- and 14-month-old rats and thereafter did not develop to the level of the other two age groups, recovering from injury by 96 h as in the 14-month-old rats. Neither hepatomicrosomal CYP2E1 protein nor the associated p-nitrophenol hydroxylase activity or covalent binding of 14CCl4-derived radiolabel to liver tissue differed between the age groups or diet regimens 2 h after the administration of 14CCl4. Compensatory liver tissue repair (3H-T, PCNA) was prompt and robust soon after CCl4 liver injury in the 14- and 24-month-old rats. In stark contrast, in the 3-month-old rats it failed allowing unabated progression of liver injury. These findings suggest that stimulation of early onset and robust liver tissue repair rescue the 14- and 24-month-old F344 rats from the lethal effect of the CD+CCl4 combination.     

Role of hepatocellular regeneration in CCl4 autoprotection

The destruction of liver microsomal cytochromes P450 by a previously administered low dose of CCl4 has been widely accepted as the mechanism of CCl4 autoprotection. However, circumstantial evidence suggests that this mechanism cannot completely explain the phenomenon of autoprotection. The protective effect of a low dose of CCl4 (0.3 ml/kg, po) on the lethal effect of a subsequently administered high dose (5 ml/kg, po) was established in male Sprague Dawley rats. The protective dose permitted 100% survival, whereas only 15% survival was observed without it. Hepatotoxicity, measured by serum enzyme elevations (aspartate transaminase, alanine transaminase, and sorbitol dehydrogenase) and histopathological changes 24 hr after the treatment with high dose, was similar in both the groups, even though the protective dose had significantly decreased liver microsomal cytochromes P450 (to 62% of normal) and associated enzymes, aminopyrine demethylase and aniline hydroxylase. Rats pretreated with CoCl2 to decrease hepatic microsomal cytochrome P450 to 44% of normal levels did not show a significant protection from the hepatotoxicity of high dose of CCl4. Previous studies have established that hepatocellular regeneration is stimulated within 6 hr after the administration of a low dose of CCl4. Based on this observation, a premise that autoprotection results from augmented recovery from injury rather than decreased injury appears likely. Hence, the role of hepatocellular regeneration was evaluated by following 3H-thymidine incorporation in hepatocellular nuclear DNA, labelling index by autoradiography, and by morphometric estimation of mitotic index. After administration of the protective dose of CCl4, stimulated nuclear DNA synthesis measured by 3H-thymidine incorporation into nuclear DNA was increased and this remained high even after subsequent administration of high dose of CCl4. Forty-eight hr after the administration of a lethal dose of CCl4 alone (5 ml/kg, po), labelling index was slightly increased, but mitotic index was not increased. In the surviving rats (15%), both labelling index and mitotic index were significantly elevated after an additional 24 hr. In rats receiving the protective dose, a significantly greater elevation of labelling index as well as mitotic index occurred 48 hr after the administration of the same lethal dose of CCl4. These results suggest that hepatocellular regeneration stimulated by the protective dose, as a biological response recruited to overcome the accompanying limited injury, may augment and sustain tissue repair processes to permit tissue restoration even after the massive liver injury elicited by the subsequent large dose of CC14.     

Treatment of chlordecone (Kepone) toxicity with cholestyramine. Results of a controlled clinical trial.

Industrial workers exposed to the organochlorine pesticide, chlordecone (Kepone), had signs of toxicity in several organs. The extent of toxicity was proportional to the levels of this chemical in the tissues. In 22 patients, chlordecone was eliminated slowly from blood (half time of 165 +/- 27 days--mean +/- S.E.M.) and fat (half time of 125 days, with a range of 97 to 177), chiefly in the stool. Output of chlordecone in bile was 10 to 20 times greater than in stool, suggesting that chlordecone is reabsorbed in the "ntestine. Cholestyramine, an anion-exchange resin that binds chlordecone, increased its fecal excretion by seven times. In a five-month trial, cholestyramine significantly accelerated elimination of chlordecone from blood, with a half life of 80 +/- 4 days (S.E.M.) (P less than 0.005) and fat (half life of 64 days, with a range of 52 to 85) (P less than 0.05). Cholestyramine offers a practical means for detoxification of persons exposed to chlordecone and possibly to other lipophilic toxins.     

Release of leukotriene B4 from human neutrophils and its relationship to degranulation induced by N-formyl-methionyl-leucyl-phenylalanine,serum-treated zymosan and the ionophore A23187.

The release of leukotriene B4 (LTB4) from human neutrophils and its relationship to degranulation induced by the divalent cation ionophore A23187, serum-treated zymosan (STZ), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and arachidonic acid (AA) have been studied. Greatest release of LTB4, measured by specific radioimmunoassay, occurred in response to A23187 (5-10 ng/10(6) cells); lower concentrations were obtained after incubation with STZ (0.2-0.8 ng/10(6) cells) and AA (0.3-2.6 ng/10(6) cells) and low (0.02 ng/10(6) cells) or not detectable amounts from cells incubated with FMLP. Release of LTB4 induced by STZ, FMLP and submaximal concentrations of A23187 was potentiated by simultaneous addition of AA. Lower amounts (0.06-0.3 ng/10(6) cells) of thromboxane B2 (TXB2) were also released by these stimuli, however this release of TXB2 was not potentiated by exogenous AA. The secretion of beta-glucuronidase induced by A23187, STZ and FMLP was not quantitatively related to release of LTB4 or TXB2 and was not potentiated by exogenous AA. Furthermore, FMLP induced degranulation was cytochalasin B (Cyt B)-dependent, whereas LTB4 release in response to this stimulus was only marginally increased by pretreatment of the cells with Cyt B. These data indicate that LTB4 does not mediate degranulation induced by these stimuli.     

Protective effect of tryptophan and cysteine against carbon tetrachloride-induced liver injury

The effects of the administration of tryptophan and/or cysteine on carbon tetrachloride (CCl4)-induced hepatic injury were investigated. Rats received CCl4 (1 ml/kg ip) followed 6 hr later by tryptophan (300 mg/kg) and/or cysteine (950 mg/kg) via stomach tube and rats were killed after 24 hr. Treatment with tryptophan, cysteine, or both reduced the degree of hepatic necrosis observed histologically. While CCl4 caused polyribosomal disaggregation and decreased [14C]leucine incorporation into liver proteins in vitro and in vivo, treatment with tryptophan, cysteine, or both caused a shift in polyribosomes toward heavier aggregation and protein synthesis was increased. Serum activities of lactic dehydrogenase (LDH), glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and gamma-glutamyl-transpeptidase were markedly increased after CCl4 alone but after subsequent treatment with cysteine or with tryptophan and cysteine appreciable decreases occurred. Glutathione concentration decreased but total amount remained constant in the livers of CCl4-treated rats while subsequent treatment with cysteine alone or together with tryptophan elevated both levels of glutathione. Using isolated hepatocytes, CCl4 caused decreases in cell viability, in release of LDH, and in [14C]leucine incorporation into protein. Treatment with CCl4 and tryptophan and/or cysteine revealed that cysteine alone or with tryptophan improved cell viability and decreased LDH release of the cells, while tryptophan alone or with cysteine improved protein synthesis. Upon cytologic evaluation, the isolated hepatocytes revealed membrane distortions after CCl4 alone but these were less marked after CCl4 plus tryptophan, cysteine, or both (most improvement). Thus, tryptophan and cysteine act in a beneficial manner against CCl4-induced hepatic injury in the rat.     

Amplification of chloroform hepatotoxicity and lethality by dietary chlordecone (kepone) in mice

Male Swiss Webster mice (25-30 g) maintained on powdered control diet, or on diets containing chlordecone (CD, 10 ppm), mirex (M, 10 ppm), or phenobarbital (PB, 225 ppm) were used in this study. At these low levels, chlorinated hydrocarbon pesticides are not toxic, they neither affect food or water consumption, nor the body weight of mice. After a 15-day dietary protocol, a single challenge dose of CHCl3 (0.1 ml/kg) was administered intraperitoneally in corn oil vehicle. Liver damage was assessed 24 hours later using serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, histopathology, and lethality. For comparison, serum enzymes were measured in a separate group of mice receiving a high dose of CHCl3 (1.0 ml/kg) alone. None of the dietary treatments alone affected any of the serum transaminases. The serum enzymes were remarkably elevated in the mice treated with CD and CHCl3. A high dose of CHCl3 (1.0 ml/kg) elevated the serum enzymes more than 10-fold over those in the mice fed normal diet receiving only the corn oil vehicle. The histopathology of the liver indicated midzonal necrosis typical of liver injury from CHCl3 and depletion of PAS positive glycogen deposits. These effects were not evident in mice treated with 0.1 ml/kg CHCl3 alone. Additional histological alterations in the livers of the CD + CHCl3 group include the degenerated cells, loss of basophilic staining characteristics, and an increased degree of cytoplasmic vacuolation. The amplification of CHCl3 hepatotoxicity by CD was also reflected by a 4.2-fold increase in lethality determined by 48-hour LD50.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of hypoxia and carbon tetrachloride toxicity in hepatocyte monolayers

The toxicity of carbon tetrachloride (CCl4) in monolayer cultures of primary hepatocytes was investigated at oxygen concentrations that prevail in the liver under conditions that range from normoxia to hypoxia: 0.5, 1, 2, and 20% O2. CCl4 was administered in the vapor phase at concentrations that produce aqueous concentrations at 37 degrees C of 0.4, 2.0, and 4.0 mM. Damage was assayed by leakage of aspartate transaminase and the inclusion of Trypan Blue immediately after the 2-hr incubation and after an additional 6-hr incubation in 20% O2. Only in the case of 0.5% O2 and 4 mM CCl4 were the monolayers damaged (18%) immediately after the 2-hr exposure; all other exposed cells were undamaged at that time point and the dose response of cell death as a function of CCl4 and oxygen concentration was not evident until the 6-hr time point. The monolayers exposed to 4 mM CCl4 and 1, 2, or 20% O2 exhibited little immediate damage but were all 100% dead 6 hr later. The monolayers exposed to 2 mM CCl4 and 0.5, 1, 2, or 20% O2 were 53, 48, 40, and 22 +/- 2% dead after 6 hr, respectively. These results suggest that effects of CCl4 exposure, for example alterations in the function or synthesis of essential proteins, require several hours to affect cell viability.     

Dependence of the carbon-tetrachloride--induced death of cultured hepatocytes on the extracellular calcium concentration.

The role of extracellular Ca2+ ions in the killing of liver cells by CCl4 was studied in primary cultures of rat hepatocytes. The dependence of in vitro cell killing on the metabolism of CCl4 was first examined in order to document the similarity between the action of CCl4 on cultured hepatocytes and the action of CCl4 on liver cells in the intact animal. Cells prepared from male rats pretreated with phenobarbital were more sensitive to CCl4 than cells prepared from either male or female rats. The killing of hepatocytes by CCl4 was prevented by addition of SKF 525A to the culture medium. This protection was accompanied by evidence of decreased CCl4 metabolism as assessed by the extent of covalent binding of 14C-CCl4 metabolites to total cellular lipids and proteins, and by the extent of formation of conjugated dienes accompanying the peroxidation of phospholipids isolated from total cell lipids. The extent of killing of the hepatocytes by CCl4 was dependent on the Ca2+ concentration in the tissue culture medium. Total Ca2+ concentrations lower than 0.10 mM were not associated with any CCl4-induced cell death, and the number of dead cells increased with increasing Ca2+ from 0.3 to 3.6 mM. This dependency on extracellular Ca2+ was not due to dependency of the extent of metabolism of CCl4 on Ca2+. The Ca2+ concentration in the medium had no effect on the extent of covalent binding of metabolites of CCl4 to lipids and to proteins and on the extent of peroxidation of phospholipids as shown by the formation of conjugated dienes. In addition, hepatocytes incubated in low Ca2+ with CCl4 developed further evidence of cell injury, as indicated by the killing of these cells following the addition of high Ca2+ concentrations under conditions prohibiting any further metabolism of the CCl4. The results of this study indicate that it is the presence of extracellular Ca2+ that converts initially nonlethal cell injury into irreversible cell injury in CCl4-treated cells. This action of Ca2+ most likely represents an influx into the cell across an injured permeability barrier at the plasma membrane, in accord with the accumulation of large quantities of Ca2+ in CCl4-intoxicated liver cells in the intact animal. The relation between this alteration in Ca2+ homeostasis and the metabolism of CCl4 is discussed.     

Alterations in hepatocyte plasma membrane in carbon tetrachloride poisoning. Freeze-fracture analysis of gap junction and electron spin resonance analysis of lipid fluidity

The plasmalemma of the livers of rats treated with carbon tetrachloride (CCl4) were examined by freeze-fracture and electron spin resonance probe techniques. The rodents received by mouth either mineral oil alone (0 to 4.5 hours before sacrifice) or CCl4 in mineral oil (1:1) (2.5 ml of CCl4/kg, 0 to 3 hours before sacrifice). Rats were anesthetized with ether and livers were perfused in situ with saline either at ambient temperature or at 4 degrees C. After perfusion, livers were fixed in situ and processed for freeze-fracture and electron microscopy. Hepatocytes were isolated and incubated with 12-doxylstearic acid and subjected to electron spin resonance analysis. Electron microscopy revealed greater than a 2.5-fold increase in the individual mean gap junction size when rats were treated with mineral oil alone for 4.5 hours and the livers were processed at room temperature. The mean gap junction size in rats dosed with CCl4 for 0.5 hours before sacrifice equalled those of the group treated with mineral oil for 4.5 hours. Increases in gap junction size with CCl4 were progressive with time; by 3 hours, a 3.5-fold increase over controls was observed (p less than 0.05). When livers were perfused with iced saline, rats treated with mineral oil for 1.5 hours had a slight decrease (not significant) in mean gap junction size as compared to controls, while the size in rats treated for the same amount of time with CCl4 increased almost 5-fold over controls (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effect of chlordecone and mirex on steroid synthesis in Y-1 cells

Chlordecone is known to bring about alterations in the endocrine systems of a variety of animals. The present study was undertaken to investigate the possibility of a direct effect of the insecticide on steroid-secreting tissues. Technical grade chlordecone produced a dose-dependent inhibition of steroidogenesis in cultured mouse adrenal tumor cells (Y-1 cells) when stimulated with ACTH (IC50 = 4 X 10(-5) M), cAMP (IC50 = 2.3 X 10(-5) M), or pregnenolone (IC50 3.5 X 10(-5) M). Similar values were obtained with 99% pure chlordecone. These data suggest that chlordecone inhibited the conversion of pregnenolone into 20 alpha-dihydroprogesterone. Additional sites of action cannot be ruled out. Mirex failed to inhibit steroidogenesis which was supported by exogenous pregnenolone. At a concentration of 3 X 10(-6) M mirex inhibited cAMP induced steroidogenesis, but higher concentrations did not produce a more pronounced effect.     

Protection of hepatotoxic and lethal effects of CCl4 by partial hepatectomy

CCl4 is a hepatotoxic haloalkane, capable of producing hepatocellular fatty degeneration and centrilobular necrosis. Previous reports indicate induction of liver regeneration after 36-48 hr of CCl4 treatment, which is considered as a secondary effect. The present investigation was undertaken to evaluate the primary effects of CCl4 on hepatic DNA synthesis and to correlate liver regeneration with CCl4 toxicity. These studies were conducted in normal and actively regenerating livers using male Sprague-Dawley rats undergoing sham operation (SH), or partial (70%) hepatectomy (PH). Incorporation of 3H-thymidine (3H-T) in hepatocellular nuclear DNA and autoradiographic analyses of liver sections served as indices for hepatocellular regeneration. Initial experiments established that peak regeneration occurs at 2 days post-PH (PH2) and liver regeneration phases out by 7 days post-PH (PH7). SH and PH rats were challenged with a single ip dose of either corn oil vehicle or CCl4 at either 0.1 ml/kg (to represent subtoxic dose) or 2.5 ml/kg (to represent toxic dose). The low dose of CCl4 was not toxic and did not alter 3H-T incorporation and percentage labelled cells at 6 or 24 hours after administration to SH, PH2 or PH7 groups, indicating that there was no interference with PH-stimulated hepatocellular regeneration. The high dose of CCl4 was significantly hepatotoxic and lethal in SH rats, while in PH2 rats both hepatotoxic and lethal effects were significantly decreased. 3H-T incorporation as well as percentage labelled cells, highly stimulated by PH, were significantly decreased by high dose of CCl4. However, hepatocellular regeneration in PH2 rats treated with high dose of CCl4 was still significantly higher than SH or PH7 groups by virtue of the stronger stimulatory effect of PH. In PH7 rats, where hepatocellular regeneration had returned to the SH level, the hepatotoxic and lethal effects of the large dose of CCl4 were also restored. These findings show that the progressive phase of a single high dose of CCl4 injury which normally culminates in hepatotoxic and lethal effects is significantly mitigated by previously stimulated hepatocellular regeneration. High dose of CCl4 suppresses hepatocellular regeneration at early time points after administration in contrast to the smaller subtoxic dose of CCl4. By virtue of the much stronger stimulatory effect, PH results in the protection against the hepatotoxic and lethal effects of CCl4 despite the obtunding effects of the high dose on hepatocellular regeneration.     

Characteristics of renin release from isolated superfused glomeruli in vitro.

1. A method is described for studying renin release from superfused rat glomeruli following their rapid isolation by a magnetic iron-oxide technique. 2. Microscopically selected glomeruli were free of tubular components. Some possessed vascular pole protrusions of up to 20 mum, unrelated to renin content. 3. Renin content of 102 batches, each of 400 glomeruli, was 1.34 plus or minus 0.08 times 10-4 Goldblatt hog units per 100 glomeruli (plus or minus S.E. of mean). Different osmolarities (305, 355 and 400 m-osmole/1.), sodium concentrations (110 and 135 mM) and buffer compositions of the preparation solution did not alter this value. Renin content per glomerulus in intact kidney was 100-fold higher. 4. At 30 degrees C the contained juxtaglomerular cells released renin at consistent but decreasing rates over 4-6 hr. Initial release rate in 110 mM sodium, 305 m-osmole/1. solutions were 0.86 plus or minus 0.068 times 10-6 units per 100 glomeruli per 30 min (plus or minus S.E. of mean, n = 42) or 0.546 plus or minus 0.046 percent of content per 30 min. In 135 mM sodium, 305 m-osmole/1. solutions, release was 2.4-fold higher (P less than 0.001) and remained elevated for at least 3 hr. When related to renin content per glomerulus resting release rate in vitro was higher by at most one order of magnitude than calculated in vivo values. 5. Release was augmented by gentle physical agitation of the glomeruli. 6. Release rate was inversely ralated to temperature. On reducing temperature from 30 degrees C, release increased 2.6-fold at 20 degrees C and 6.7-fold at 10 degrees C (P less than 0.001, n = 11). The response was reversible. 7. 3 mM sodium cyanide plus 3 mM sodium iodoacetate caused a variable release of renin associated with depletion of content within 4 hr. The response was progressive and reached a peak after 60 min. 8. Sensitivity of renin release to temperature and metabolic blockade indicates that energy is required for retention of renin by the cell. This, together with the release observed with increased sodium concentration at constant osmolarity, suggests a dependence of renin release upon the mechanism controlling the volume of the juxtaglomerular cell or its organelles.     

Studies on renin release from isolated superfused glomeruli: effects of temperature, urea, ouabain and ethacrynic acid.

1. The effects of different energy substrates, of low temperature, of urea, and of ouabain and ethacrynic acid were studied on the rate of renin release from viable juxtaglomerular cells during superfusion of isolated rat glomeruli. 2. Neither lactate nor glutamate altered renin release rate from that observed using glucose as the sole energy substrate. Succinate 10 mM elevated release transiently but did not influence the release caused by reductions in osmolality through lowering sucrose concentration. 3. Peak renin release was more prolonged and returned more slowly to control following reductions in osmolality in phosphate-Ringer than in bicarbonate-Ringer. 4. At 37 degrees C, the peak of renin released induced by hypo-osmolality was smaller and delayed, and returned earlier to control than at 30 degrees C. Reduction in temperature from 30 to 4 degrees C resulted in a 32-fold increase in basal release rate. At 4 degrees C a 20 m-osmole/kg reduction in tonicity caused an additional 2-5-fold increase in release rate. 6. Increasing superfusate osmolality with urea did not affect basal renin release but 100 mM urea suppressed the releasing effect of a 15 mM reduction in NaCl concentration. 7. Ouabain (10(-4) M) caused a small (33 +/- 9%, P less than 0-025) transient increase in renin release. Ethacrynic acid (10(-3) M) provoked a progressive increase in release reaching 100 +/- 15% above control within 50 min. In the presence of both inhibitors the release provoked by hyposmolality was prolonged. 8. It is concluded that renin release in vitro is a function of actively regulated cell volume and it is proposed that a similar mechanism could underline both barorecptor and macula densa controls of renin secretion in vivo.     

Hypotonicity-induced ATP release is potentiated by intracellular Ca2+ and cyclic AMP in cultured human bronchial cells

We have examined the cultured human bronchial epithelial cells (16HBE) to learn if changes in Cl(-) concentration or osmolality stimulate the cells to release ATP and to determine whether its release is cyclic AMP (cAMP)- and/or Ca(2+)-dependent by using the luciferin-luciferase luminometric assay. In a control solution (290 mosmol kg H(2)O(-1)), the external ATP concentration and the rate of ATP release were 0.52 +/- 0.20 nM and 0.036 +/- 0.034 pmol min(-1), respectively. Upon hypotonicity (205 mosmol kg H(2)O(-1)), they increased to 7.0 +/- 1.3 nM and 3.1 +/- 0.6 pmol min(-1), respectively, at 6 min, then decreased. At the peak, the rate of ATP release is estimated to be 6.2x10(4) ATP molecules s(-1) per cell. An accumulation of the released ATP for the initial 10 min increased significantly (p < 0.005) by 71.5% in the presence of forskolin (10 microM), adenylyl cyclase activator, however, it was abolished (p < 0.001) by pretreatment with BAPTA-AM (25 microM), a membrane permeable Ca(2+) chelator. On the other hand, neither low Cl(2-) (75 mM, isotonic) nor hypertonicity (+NaCl or +mannitol, 500 mosmol kg H(2)O(-1)) could significantly increase the ATP release. Further, forskolin or ionomycin (a Ca(2+) ionophore) or, both, failed to stimulate ATP release under the isotonic condition. In conclusion, first, hypertonicity and changes in Cl(-) concentrations are not effective signals for the ATP release; second, hypotonicity-induced ATP release is potentiated by the level of intracellular Ca(2+) and cAMP; and third, a biphasic increase in ATP release and its low rate at the peak support the hypothesis that ATP is released through a non-conducting pathway model, such as exocytosis, or through a volume-dependent, ATP-conductive anion channel.