Centrilobular Hepatocytes Research Articles

Induced Cytolethality and Regenerative Cell Proliferation in the Livers and Kidneys of Male B6C3F 1 Mice Given Chloroform by Gavage

Induced Cytolethality and Regenerative Cell Proliferation in the Livers and Kidneys of Male B6C3F 1, Mice Given Chloroform by Gavage. Larson, J. L., Wolf, D.C., and Butterworth, B. E. (1994). Fundam. Appl. Toxicol. 23, 537-543. It has been reported that chloroform administered to male B6C3F 1 mice at doses of 138 and 277 mg/kg/day in corn oil by gavage 5 days/week for 2 years resulted in incidences of hepatocellular carcinomas of 36 and 98% relative to an incidence in controls of 6%. Cytotoxicity and regenerative cell proliferation have been implicated in the tumorigenic process for this nongenotoxic compound. Although chloroform is known to be nephrotoxic in the male mouse, no treatment-related increase was observed in the frequency of kidney tumors. To better understand the relationship of these endpoints, this study evaluated chloroform-induced cytotoxicity and cell proliferation in the liver and kidney under conditions of the cancer study. B6C3F 1 mice were administered oral doses of 0, 34, 90, 138, or 277 mg/kg/day of chloroform dissolved in corn oil for 4 days or 5 days/week for 3 weeks. Bromo-2′-deoxyuridine (BrdU) was administered via osmotic pumps implanted 3.5 days prior to necropsy to label cells in S-phase. Cell proliferation was evaluated in tissue sections immunohistochemically as the percentage of cells in S-phase (nuclear labeling index; LI). Mice given 34 and 90 mg/kg/day by gavage had mild degenerative changes in centrilobular hepatocytes after 4 days of treatment, which was absent at 3 weeks. Centrilobular necrosis was observed in mice given 138 or 277 mg/kg chloroform for 4 days, with increased severity of necrosis at 3 weeks. The LI in the livers of mice was increased in a dose-dependent manner at all chloroform doses following 4 days of treatment. The hepatic LI was similarly elevated at 3 weeks in the 277-mg/kg dose group, but had declined in the lower dose groups, and was no longer elevated above controls at 34 or 90 mg/kg/day. Kidneys from mice in all dose groups exhibited a dose-related acute tubular necrosis after 4 days of dosing. After 3 weeks of dosing, regenerating tubules were observed in the lower dose groups, while mice treated with 277 mg/kg had severe nephropathy characterized by degeneration, necrosis, and regeneration affecting all of the proximal tubules. The LI in the proximal tubules was increased at all doses after 4 days of treatment. At the end of 3 weeks of dosing, the LI in the renal cortex had decreased at all dose levels and was no longer significantly different from controls in the 34- and 90-mg/kg/day groups. The sustained increase in LI seen in the livers of mice administered not only the maximum tolerated dose (MTD), but also one-half the MTD is consistent with the hypothesis that events secondary to cytolethality and regenerative cell proliferation play a key role in chloroform-induced mouse liver cancer. The observation of toxicity and acute regenerative cell proliferation in the kidney without subsequent tumor formation may be because the severity of the nephrotoxicity in some way actually interfered with tumor formation. Alternatively, the mouse liver may simply be a more sensitive target organ than the kidney to tumor formation induced through mechanisms secondary to regenerative cell proliferation.

Sequential functional and morphological alterations during hepatocarcinogenesis induced in rats by feeding of a low dose of 2-acetylaminofluorene.

The early cellular events in liver carcinogenesis were studied in Fischer-344 male rats that either were fed 200 ppm 2-acetylaminofluorene (AAF) for up to 10 wk or were fed the carcinogen for 8 wk followed by maintenance for an additional 24 wk. By 1 wk of exposure, AAF caused a reduction in the number of glutamine synthetase (GS)-positive centrilobular hepatocytes, an increase in DNA synthesizing hepatocytes in the central areas of the hepatic lobules, and a shift from multinucleated to mononucleated hepatocytes, although overt hepatocellular necrosis was not evident. By 3 wk, altered hepatocellular foci characterized by deficiencies in iron storage (IS-) and collagen production and by expression of gamma-glutamyl transferase (GGT+) and placental-type glutathione transferase (PGT+) activity appeared. Single PGT+ cells were also found. During continued exposure, foci increased in number, size, and total area with the increases escalating between 8 and 10 wk of exposure. Cessation of AAF exposure at 8 wk resulted in a slight decrease in the number of foci after a further 6 wk of maintenance, but with continued maintenance for another 6 and 12 wk, the number again increased. IS- characterized the majority of foci during carcinogen administration, whereas after cessation of exposure, GGT+ and PGT+ foci predominated. None of the foci were positive for GS. After AAF exposure for 10 wk, a few neoplasms developed and greater numbers occurred after maintenance for a further 24 wk of rats exposed for 8 wk. We conclude the following: (a) the low dose of AAF caused subtle alterations in function and proliferation of normal hepatocytes and converted hepatocytes into focus cells; (b) reduction of the GS+ area is a sensitive indicator of cytotoxicity of AAF; (c) the development of some foci at an early stage depends on a promoting action of AAF, which ceased when the carcinogen was withdrawn, allowing some foci to undergo reversion; (d) a strong linkage exists in expression of IS-, GGT+, and PGT+ in foci; (e) the carcinogenic process accelerates in the absence of any indication of increased cytotoxicity by AAF; and (f) under the conditions of this study, no GS+ foci, adenomas, and carcinomas were found, indicating that no carcinogen-induced expression of GS occurred in these lesions and that GS expression is not linked to other phenotypic abnormalities.

Short-term feeding of a diet enriched in phospholipids increases bile formation and the bile acid transport maximum in rats

Earlier studies suggested that the secretory rate maximum (SRm) of bile acid and the cholestasis which occurs after the SRm is reached may be determined by the hepatic or extrahepatic biliary phospholipid pool. We therefore investigated whether bile formation and the bile acid SRm could be influenced by feeding a diet enriched in phospholipids. Male rats were fed phospholipid (PLD) or triacylglycerol (TgD) -enriched diet for 3 days, and bile formation as well as biliary lipid output were measured on the 4th day. In other similarly fed groups, cholic acid was infused in stepwise increasing doses to determine the effect of PLD on the SRm of cholic acid. The plasma lipid levels were significantly lower in PLD and TgD diets compared to basal diet. But, while the levels of total cholesterol (CH), HDL-CH, and phospholipid (PH) were not significantly altered by PLD compared to TgD, the triacylglycerol levels were markedly increased by PLD. In the liver of PLD fed rats, triacylglycerol and CH ester contents decreased by 39 and 62%, respectively, while free CH and PH contents were not significantly changed. The PLD significantly augmented spontaneous bile flow, bile acid, PH and CH secretion rates compared to TgD diet (65, 124, 164 and 654%, respectively). The enhanced biliary secretory function was associated with an increase in pericanalicular vacuoles and diverticuli in centrilobular hepatocytes. Compared to TgD fed rats, PLD rats showed a 2-fold decrease in the ratio of cholic acid/chenodeoxycholic acid in bile and a significant decrease in the % contribution of taurine conjugated BA. The PH fatty acids in bile were similar in both groups except that in PLD group the % contribution of C18:2 was higher than in TgD group. No differences were found in plasma membrane CH/PH content or total fatty acid composition. During bile acid infusion, the SRm and the total cholic acid secreted were significantly higher in the PLD than in the TgD rats. Moreover, the cholestatic response observed after high bile acid dose was markedly reduced by PLD. The results show that short-term feeding of PLD induces changes in CH and bile acid metabolism which result in enhanced biliary output of CH and PH. The enhanced pool of biliary lipid may protect plasma membranes from the deleterious effects of high bile acid concentrations.

Recombinant insulin-like growth factor-II inhibits the growth-stimulating effect of growth hormone on the liver of Snell dwarf mice.

The actions and interactions of recombinant insulin-like growth factor-I and -II (IGF-I and IGF-II), alone or in combination with human GH on body growth and the growth of several organs were studied in the Snell dwarf mouse. IGF-I and -II stimulate to a similar extent sulfate incorporation into cartilage, and both IGFs increase body length and weight. IGF-II as well as IGF-I have clear effects on the size of the submandibular salivary glands, kidneys, and spleen. IGF-II, however, did not influence the weight of the lung, in contrast with IGF-I. GH treatment alone resulted in growth of the liver, whereas both IGFs were inactive. Surprisingly, IGF-II and, to a lesser extent, IGF-I inhibited GH-induced growth of the liver. Glycogen storage in the liver was decreased by treatment with IGF-II alone or in combination with GH, as shown by histological examination. It was not affected by GH, IGF-I, or GH plus IGF-I. Also, the size of the centrilobular hepatocytes was decreased by treatment with IGF-II and IGF-II plus GH; GH alone had a hypertrophic effect, whereas IGF-I or GH plus IGF-I had none. In contrast to GH, IGFs did not increase polyploidy. Treatment with IGF-II increased the level of IGFBP-3, as did IGF-I or GH treatment, as shown by Western ligand blotting. The IGFs appeared to have a greater effect on the induction of 38.5-kilodalton IGFBP-3 than GH, suggesting a different role in the regulation of glycosylation. In conclusion, IGF-I and IGF-II as well as GH have a stimulatory effect on general body growth and are effective in the stimulation of serum IGFBP-3, sulfate incorporation into cartilage, as well as the growth of specific organs in Snell dwarf mice. Both IGFs, alone or in combination with GH, show distinct effects on the growth of the liver with respect to several histological parameters, which require further exploration.

The Toxicity of 1-Week Exposures to Inhaled Chloroform in Female B6C3F1 Mice and Male F-344 Rats

The Toxicity of 1-Week Exposures to Inhaled Chloroform in Female B6C3F1 Mice and Male F-344 Rats. Larson, J. L., Wolf, D. C., Morgan, K. T., Mery, S., and Butterworth, B. E. (1994). Fundam. Appl. Toxicol. 22, 431-446.Detailed quantitative descriptions of the toxicity of inhaled chloroform are lacking, despite the fact that the majority of environmental exposures occur by this route. We investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the livers and kidneys, the principal target organs for carcinogenicity of female B6C3F1 mice and male F-344 rats, respectively. Nasal passages were also examined for toxic responses. Rodents were exposed to chloroform vapors at concentrations of 0, 1, 3, 10, 30, 100, or 300 ppm for 6 hr/day for 7 consecutive days and necropsied on Day 8. Animals were administered bromodeoxyuridine (BrdU) via implanted osmotic pump for the previous 3.5 days before necropsy. Cell proliferation was quantitated as the percentage of cells in S-phase (labeling index; LI) measured by immunohistochemical detection of BrdU-labeled nuclei. Mice exposed to 100 or 300 ppm exhibited centrilobular hepatocyte necrosis and severe vacuolar degeneration of midzonal and periportal hepatocytes, while exposure to 10 or 30 ppm resulted in mild to moderate vacuolar changes in centrilobular hepatocytes. Slight, dose-related increases in the hepatocyte LI were observed for exposure concentrations of 10 and 30 ppm, while the LI was increased more than 30-fold in the 100 and 300 ppm groups. The kidneys of mice were affected only at the 300 ppm exposure, with approximately half of the proximal tubules lined by regenerating epithelium and an increased LI of tubule cells of 8-fold over control. In rats, mild centrilobular vacuolation was observed only in the livers of rats exposed to 300 ppm. The hepatocyte LI in rats were increased only at 100 and 300 ppm, 3- and 7-fold over control, respectively. In the kidneys of the male rats exposed to 300 ppm, about 25 to 50% of the proximal tubules were lined by regenerating epithelium. The LI for tubule cells in the cortex was increased at 30 ppm and above. In the nasal passages of rats, chloroform concentrations of 10 ppm and above induced histopathological changes that exhibited clear concentration-related severity. These lesions consisted of respiratory epithelial goblet cell hyperplasia and degeneration of Bowman's glands in olfactory mucosa with an associated osseous hyperplasia of the endoand ectoturbinates in the periphery of the ethmoid region. These nasal lesions were not observed in mice. Knowledge of the dose-dependent responses in rats and mice will be valuable in assessing the potential risks to humans posed by inhaled chloroform and in setting exposure concentrations for longer-term studies.

The Interactive Toxicity of CHCI3 and BrCCI3 in Precision-Cut Rat Liver Slices

The Interactive Toxicity of CHCI3 and BrCCI3 in Precision-Cut Rat Liver Slices. Azri-Meehan, S., Mata, H. P., Gandolfi, A. J., and Brendel, K. (1994). Fundam. Appl. Toxicol. 22, 172-177.The interactive toxicity of two nontoxic concentrations of chloroform (CHCI3) and bromotrichloromethane (BrCCI3) 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 CHCI3 or 0.1 125 μl (0.25 mg, 1.26 μmol) BrCCI3 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 BrCCI3, alone or when added with CHCI3. This loss may be attributed to BrCCI3-induced suicide inactivation of cytochrome P450. Centrilobular hepatocytes may be more susceptible to the interactive toxicity of CHCI3 and BrCCI3. 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 BrCCI3 and CHCI3 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 BrCCI3 and CHCI3.

Use of precision-cut liver slices for studies of unscheduled DNA synthesis

Precision-cut liver slices were prepared from untreated and Aroclor 1254 (ARO)-treated male Sprague-Dawley rats with a Krumdieck tissue slicer. Liver slices were cultured for 24 hr in medium containing [ 3H]thymidine and 0–0.1 m m 2-acetylaminofluorene (2-AAF) using a dynamic organ culture system and processed for autoradiographic evaluation of unscheduled DNA synthesis (UDS). Compared with control (i.e. 0 m m 2-AAF) liver slice cultures, 2-AAF produced a concentration-dependent increase in UDS, the effect being more marked in liver slices from ARO-treated than from untreated rats. With liver slices from untreated rats, 2-AAF produced the greatest increase in UDS in centrilobular hepatocytes. 2-AAF-induced UDS in liver slices from ARO-treated rats was most marked in centrilobular hepatocytes but the effect also extended to other areas of the liver lobule. These results demonstrate that precision-cut liver slices may be a valuable alternative in vitro system to hepatocyte cultures for screening chemicals for potential genotoxicity. Unlike hepatocyte cultures, liver slices permit the study of zonal differences in UDS. Moreover, this technique could be applied to other tissues and the study of species differences in response.

Immunohistochemical and microfluorometric determination of hepatic DNA adduct removal in rats fed 2-acetylaminofluorene.

Biphasic removal of DNA adducts has previously been demonstrated by radioimmunoassay in whole liver DNA from rats chronically fed 2-acetylaminofluorene for 28 days. In the present study, removal of N-(deoxyguanosin-8-yl)-2-aminofluorene was observed in situ by microfluorometry. Frozen liver sections from animals fed 0.02% 2-acetylaminofluorene for 28 days, followed by a control diet for 3, 7, 14, 21 and 28 days, were examined immunohistochemically for localization of N-(deoxyguanosin-8-yl)-2-aminofluorene with fluorescein-conjugated secondary antiserum. In addition, bile ducts and oval cells were stained with antibodies to keratins using Texas red-labeled indirect immunofluorescence. Hoechst dye was used to identify DNA in nuclei. During the 28 days on the control diet, after 28 days of feeding 2-acetylaminofluorene, the DNA adduct concentrations of parenchymal liver cells were reduced by 85%, as compared to animals fed only the carcinogen for 28 days. Periportal hepatocytes exhibited biphasic (fast and slow) adduct removal. Only fast adduct removal was demonstrated in midzonal and centrilobular hepatocytes, since the adduct levels were below the detectable range in these regions after 7 days on the control diet. After 28 days on the control diet, N-(deoxyguanosin-8-yl)-2-aminofluorene was detected in approximately 50% of periportal hepatocytes. These results are compatible with the previously observed biphasic removal profile determined by radioimmunoassay of whole liver DNA adducts and indicate that periportal hepatocytes remove adducts from two distinct genomic compartments.

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.

Comparison of the hepatic effects of coumarin, 3,4-Dimethylcoumarin, dihydrocoumarin and 6-methylcoumarin in the rat

The mechanism of coumarin-induced hepatotoxicity in the rat has been investigated by comparing the effects of coumarin with those of three coumarin derivatives, namely 3,4-dihydrocoumarin (DHC), 3,4-dimethylcoumarin (3,4-DMC) and 6-methylcoumarin (6-MC). Male Sprague-Dawley rats were fed either control diet or diets containing 0.5 or 0.75% coumarin, 0.76% DHC, 0.6 or 0.9% 3,4-DMC or 0.82% 6-MC for 13 wk. The dietary levels of 0.5% coumarin and 0.6% 3,4-DMC, were equimolar (3.43mmol/100g diet), as were the dietary levels of 0.75% coumarin, 0.76% DHC, 0.9% 3,4-DMC and 0.82% 6-MC (5.14mmol/100g diet). All treatments resulted in an increase in relative liver weight, but only coumarin increased plasma alanine aminotransferase and aspartate aminotransferase activities. Morphological examination of liver sections from coumarin treated rats revealed vacuolation of centrilobular hepatocytes and bile duct hyperplasia. Cholangiofibrosis was also observed, particularly in rats given 0.75% coumarin. Treatment with DHC produced no abnormalities, whereas a slight hypertrophy of centrilobular hepatocytes was observed in some 3,4-DMC treated animals and a slight vacuolation of individual hepatocytes was noted in some 6-MC treated rats. DHC, 6-MC and particularly 3,4-DMC treatment resulted in an induction of cytochrome P-450 dependent mixed function oxidase enzyme activities. All treatments induced hepatic GSHS-transferase and γ-glutamyltransferase activities, induction being most marked in rats given coumarin and 6-MC. These results provide further evidence that coumarin-induced hepatotoxicity in the rat is due to the formation of a 3,4-epoxide intermediate.

DEPRESSIVE EFFECT OF T-2 TOXIN ON HEPATOCYTE PROLIFERATION UNDER TWO EXPERIMENTAL CONDITIONS

The effect of T-2 mycotoxin on hepatocyte proliferation was investigated in mice using two models of hepatocyte regeneration, i.e. 2/3 partial hepatectomy (Ph) and CCl4-intoxication (CCl4). At 2 days after Ph or CCl4, mice were given T-2 toxin (2 or 4mg/kg, po). Five mice each were killed daily up to 7days after Ph or CCl4, and they were injected with BrdU (100mg/kg, ip) at I hour before sacrifice. T-2 toxin significantly delayed the recovery of liver weight loss after Ph Or CCl4, and it also significantly delayed the reduction in size of centrilobular hepatocyte necrosis after CCl4. In addition, T-2 toxin significantly depressed BrdU-uptake by hepatocytes in the early days after Ph or CCl4. It can be said that T-2 toxin depressed the proliferation of hepatocytes, resulting in the delay of recovery of liver weight loss. This seems to be related to the inhibitory effect of T-2 toxin on nuclear acid synthesis of hepatocytes.

Association between responsiveness to phenobarbital induction of CYP2B1/2 and 3A1 in rat hepatic hyperplastic nodules and their zonal origin.

To explore further the mechanism underlying the alteration in expression of P450 enzymes in hepatic preneoplastic lesions, expression of CYP2B1/2 and 3A1 in individual hepatic hyperplastic nodules induced by an aflatoxin B1 (AFB) administration protocol and the Solt-Farber resistance protocol in male F344 rats was examined via immunohistology. In nodules induced by the resistance protocol, expression of both CYP2B1/2 and 3A1 proteins was highly variable among different nodules, whereas these P450 proteins were expressed in all nodules induced by the AFB protocol. Nodules induced by the resistance protocol have been shown previously to arise from throughout the acinar lobule. In contrast to the resistance protocol, the AFB protocol causes extensive periportal necrosis, potentially resulting in a heavy selection pressure for clonal expansion of initiated cells arising from the centrilobular area. As phenobarbital-induced expression of both CYP2B1/2 and 3A1 in normal liver is heavily localized to the centrilobular zone, these observations suggest that the ability of preneoplastic nodules to respond to induction of these P450 proteins is determined primarily from the zonal origin of the precursor hepatocytes. Thus, the nodules from the resistance protocol that express little or no CYP2B1/2 and 3A1 may have been derived from the periportal hepatocytes, whereas all the nodules in the AFB group and some of the nodules from the resistance protocol which expressed these P450 proteins in response to phenobarbital induction may have been derived from centrilobular hepatocytes.

Pathogenesis of centrilobular necrosis following congestion of the liver.

The exact pathogenesis of centrilobular necrosis following congestion of the liver is still unknown. We reviewed the clinical data related to systemic circulatory disturbance and histopathology of the liver and the gut in 320 autopsy subjects. Congestion of the liver alone was associated only with atrophy and loss of hepatocytes in centrilobular areas, but not with hepatocellular coagulative necrosis. In many patients with coagulative necrosis of centrilobular hepatocytes and congestion of the liver, fibrin thrombi and neutrophil infiltration in the sinusoids, which are the characteristic histopathological features of the liver in endotoxaemia, were found in and around the necrotic area. Congestion, erosion or haemorrhage of the intestinal mucosa, which may allow entrance of endotoxin into the liver through the portal vein, was seen in such patients. Prolonged hypotension or shock, which may lead to portal endotoxaemia, was present in half the patients with centrilobular necrosis and congestion of the liver. These results suggest that not only congestion of the liver but also portal endotoxaemia may be involved in the pathogenesis of centrilobular necrosis in patients with congestion of the liver.

Halothane-induced cytotoxicity to rat centrilobular hepatocytes in primary culture is not increased under low oxygen concentration.

Halothane can be metabolized by both oxidative and reductive pathways in the liver. This anesthetic can induce direct liver injury preferentially localized in centrilobular areas, probably in relation with lower oxygen tension. The reductive pathway has been related to liver damage; however, a correlation between lower oxygen concentration in centrilobular areas, the extent of reductive metabolism of halothane, and the degree of liver injury has not yet been demonstrated. This study was designed to better evaluate the toxicity of the reduced metabolites by using centrilobular and periportal rat hepatocyte subpopulations. Adult rat hepatocytes, either as whole cell preparations or after separation in centrilobular and periportal cell subpopulations, were placed in primary culture and exposed to either 2% or 4% halothane under various oxygen concentrations. The enriched centrilobular hepatocyte subpopulations isolated by the digitonin-collagenase method were characterized by immunolocalization of glutamine synthetase. Three oxygen concentrations were tested: 5%, 20%, and 95%, and the main parameters measured were cell viability and fluoride ion formation. Viability of centrilobular hepatocytes was similar under 5% and 20% O2, but the unpurified hepatocyte population was more susceptible to 5% O2 (P < 0.01). Significantly higher cytochrome P-450 content was found in whole hepatocyte populations under 5% versus 20% oxygen, indicating that centrilobular hepatocytes that contained higher cytochrome P-450 monooxygenase activities were less sensitive to low oxygen concentrations. Halothane toxicity to centrilobular hepatocytes was enhanced under 95% versus 20% O2 (P < 0.05). By contrast, no significant difference was observed when the cells were maintained under 5% O2, although fluoride ions, indicative of reductive metabolism of halothane, were found in much higher amounts in the culture medium. Moreover, under 20% O2, halothane toxicity was significantly greater in centrilobular versus unpurified hepatocytes (P < 0.05). Isolated centrilobular hepatocytes appear to be more sensitive to halothane than their periportal counterparts in vitro. However, the authors' results support the conclusion that increased reductive metabolism of halothane induced by decreasing oxygen concentration is not a critical parameter for the occurrence of liver damage in these cells.

Comparative Toxicity in Rats and Dogs of Intravenous 1,3-Di(4-Imidazolino-2-Methoxyphenoxy) Propane · Lactate, a Potential Agent for the Treatment of Pneumocystis carinii Pneumonia

The subchronic toxicity of DMP · lactate [1,3-di(4-imidazolino-2-methoxyphenoxy)propane · lactate], a new agent with potential for the treatment of the opportunistic infection of Pneumocystis carinii pneumonia (PCP) in immunocompromised patients, was defined in rats and dogs after 28 (rats) or 29 (dogs) daily intravenous doses. Dose levels for both species were 0, 0.5, 2.5 and 4.0 mg/kg/day. Tremors were observed in both rats and dogs at the 2.5 and 4.0 mg/kg doses, while incontinence, anorexia, rhinorrhoea, salivation, convulsions, seizures, moribundity and death occurred in dogs at these doses. All rats survived to the scheduled sacrifice, whereas only 1 male and no female dogs administered 4.0 mg/kg/day survived to day 28 and 1 female at 2.5 mg/kg/ day was moribund on day 27. Languid behaviour was noted in rats and dogs at the highest dose. An alteration in tapetum lucidum reflection was found in dogs dosed at 2.5 and 4.0 mg/kg/day. No haematological or urinalysis indicators of toxicity were seen in either species. Serum alanine amino transferase, aspartate amino transferase and alkaline phosphatase were elevated in dogs at 2.5 and 4.0 mg/kg, while only alkaline phosphatase was increased dose-dependently in rats. No histopathological lesions were found in rats. Periportal inflammation, cytoplasmic vacuolisation of centrilobular hepatocytes and/or hepatocyte hyaline droplet formation were seen in dogs at all doses. Thus, dogs appear to be much more sensitive to DMP toxicity than rats. The differential toxicity response to DMP could be attributed to the different pharmacokinetics in the rat and the dog.

Histochemical demonstration of copper in LEC rat liver

Livers of LEC rats were histochemically stained for copper according to the modified Timm's method, which includes trichloroacetic acid (TCA) treatment. TCA pretreatment was effective in removing zinc and iron, leaving as the major metal in the liver. Hepatocytes in 3-month-old rats were stained intensely by the modified Timm's method, both in frozen sections and in paraffin-embedded specimens. The centrilobular hepatocytes were usually stained, but positive cells were also randomly distributed in the hepatic lobes, showing a mosaic pattern. The staining was intensified in 8- compared to 3-month-old LEC rats. In contrast hepatocytes from LEA rats, the normal counterpart of LEC rats, were faintly stained for copper. Proliferating cholangioles found in older LEC rats were shown to lack copper deposition, and hepatocellular carcinoma showed less copper deposits than the hepatocytes surrounding the tumor. The copper staining was augmented in livers of LEC rats subjected to copper-loading, but was less intense in the livers treated with D-penicillamine. The staining intensity under the various experimental conditions showed good correlation with the copper concentration. Lysosomal deposition of copper in hepatocytes was demonstrated by electron microscopic analysis for copper. Thus the modified Timm's method was shown to produce valuable results in demonstrating copper in LEC rat livers, providing important information for an understanding of the mechanism of copper deposition and hepatic disease of the animal.

1-Nitronaphthalene Toxicity in Rat Lung and Liver: Effects of Inhibiting and Inducing Cytochrome P450 Activity

In rats, 1-nitronaphthalene (1-NN) causes both pulmonary and hepatic toxicity. Pulmonary toxicity is evident as bronchiolar damage, with necrosis of Clara cells and ciliated cells, whereas hepatic injury involves vacuolation of centrilobular hepatocytes. Pretreatment with O, O, S-trimethylphosphorodithioate [OOS-MeP(S)] or p-xylene gave three- to fourfold protection against 1-NN toxicity. These pretreatments also prevented both the increase in lung weight and the rise in γ-glutamyltranspeptidase and alkaline phosphatase activity in bronchoalveolar lavage fluid normally associated with 1-NN toxicity. Pretreatment with Aroclor 1254 or β-naphthoflavone (β-NF) did not alter the LD50 of 1-NN. Aroclor or β-NF pretreatment did, however, prevent morphological signs of lung injury and any increase in either lung weight or enzyme activity in bronchoalveolar lavage fluid. Liver damage was not prevented by these treatments; indeed, injury was exacerbated and was transferred from centrilobular to periportal areas. In control rats the covalent binding of [1 14C]NN to liver microsomes was eight times greater than to lung microsomes. Pretreatment with OOS-MeP(S) decreased covalent binding to lung microsomes, without affecting binding to liver microsomes. Conversely, both Aroclor and β-NF slightly increased covalent binding in lung, but increased liver binding by 250-300%. Phenobarbitone also increased binding to liver microsomes by 250-300%, but failed to increase, or alter, the distribution of liver damage. The reported effects of these pretreatments indicate that the toxicity of 1-NN is probably activated by isoenzyme CYP2B1 in lung, but by isoenzymes CYP1A1 or CYP1A2 in the liver.

Relationship between genomic DNA ploidy and parameters of liver damage during necrosis and regeneration induced by thioacetamide

Thioacetamide proved to be a potent necrogenic agent when a single dose of 6.6 mmol/kg was administered intraperitoneally to rats. Its necrogenic ability was assessed on the basis of morphological and biochemical changes. The injury of centrilobular hepatocytes showed a peak of cell death 24 hr after thioacetamide administration; it was followed immediately by the regenerative response. Parallel increases of serum aminotransferases, isocitrate dehydrogenase and gamma-glutamyl transferase activities were observed. Severe liver damage was also evident at 24 hr on the basis of glutathione depletion (29% of control), malondialdehyde production (169%), cytochrome P-450 level decrease (26%) and increased activity of glutathione S-transferase (160%). We checked the regenerative response by determining nuclear DNA content in isolated hepatocytes 0, 6, 12, 18, 24, 36, 48 and 72 hr after thioacetamide administration. Changes in DNA cell distribution between G0-G1, S and G2 + M phases of the cell cycle were observed. The sharp decrease in the percentage of the tetraploid cell population (G2 + M phases) and the abrupt increase of the S-phase cells at 36 and 48 hr suggest transition from adult to fetal in hepatocyte populations obtained 24 and 36 hr after thioacetamide treatment. At 72 hr of treatment, hepatocyte populations showed recovery to adult state. In the shift from the adult to fetal, registered at 24, 36 and 48 hr after thioacetamide administration, mitosis seemed to precede the synthesis of DNA.

Dose response effects of 2-acetylaminofluorene on DNA damage, cytotoxicity, cell proliferation and neoplastic conversion in rat liver

This study measured the effect of precise doses of 2-acetylaminofluorene (AAF) in inducing DNA damage, functional changes and neoplastic conversion in rat liver. Groups of male F344 rats at 9 weeks of age were exposed to cumulative doses of 0.5 or 2.0 mmol AAF per kg body weight given by gavage daily 5 days per week over an 8-week period and maintained with no further exposure for up to 8 weeks. Administration of AAF resulted in the formation of N-deoxyguanosin-(8-yl)-2-aminofluorene in liver DNA in relationship to dose. In centrilobular hepatocytes the zone of glutamine synthetase-expressing cells was reduced by exposure. By 8 weeks, but not at 4 weeks, the higher of the two doses of AAF provoked an increase in cell proliferation measured by immunohistochemical incorporation of bromodeoxyuridine. Altered hepatocellular foci expressing the placental form of glutathione transferase were induced by the high dose of AAF at 4 weeks, but not at the low dose. At 8 weeks the incidence of foci at the high dose was 79 times that induced by the low dose. These foci were highly proliferative. In animals exposed to AAF for 8 weeks and maintained for 4 weeks with no exposure, DNA adducts decreased by 80% and cell proliferation subsided by 80%, although the glutamine synthetase zone remained diminished. After discontinuation of AAF, the number of foci diminished by 50% and their proliferation subsided by 80% at 4 weeks, indicating a phenotypic reversion of many foci. With this protocol of administration of precise doses of AAF, we have established non-linearity of effects and a lack of correlation between DNA adduct formation and induction of cellular lesions. We suggest that doses in the range of those reported can be used to study the contribution of epigenetic and genotoxic effects in carcinogenesis and to study threshold events.

Acute and Subacute Oral Toxicity of Selenocystine in Mice.

The acute and subacute oral toxicity of selenocystine was assessed in ICR male mice. In the acute study, the estimated LD50 value for a single oral dose was 76.0 mg/kg (confidence limit : 64.4-89.7 mg/kg) and the minimal lethal dose was 43.2 mg/kg. In the mice administered 50 mg/kg, the liver and kidney injuries were assumed by increase in aspartate aminotransferase, alanine aminotransferase, urea nitrogen and phosphorus, and a decrease of calcium in plasma. In the subacute study, the body weight gain of animals orally administered 10, 20, 30 and 40 mg/kg/d for 30 d significantly decreased with the dosage, and all mice given 30 or 40 mg/kg/d died within the exposure period. Although renal damage was not recognized by biochemical measurements and histopathological examinations, liver damage was apparent by increase in aspartate aminotransferase and alanine aminotransferase in plasma and histopathological findings, vacuolation of centrilobular and/or peripheral hepatocytes. Gel chromatography was carried out on the cytosol from kidney and liver of mice following a single and consecutive administrations of selenocystine. In the kidney, selenium following a single dosage of 50 mg/kg was eluted around the void volume and another fraction in Sephadex G-25 gel chromatography ; most of the selenium following consecutive dosages of 20 mg/kg/d for 30 d, however, was eluted only in the void volume. In Sephadex G-150 gel chromatography of the liver, most of the selenium following a single administration was eluted in the low-molecular fraction, whereas a major peak of selenium following consecutive administrations was eluted in the high-molecular fraction. The findings thus indicate that in the acute toxicity study selenocystine caused hepatic damage and renal damage and in the subacute toxicity liver damage, depending on selenium behavior in these organs.