Centrilobular Hepatocytes Research Articles

Studies on the mechanism of dimethylnitrosamine-induced acute liver injury in mice

Male and female adult C3H- +/+, C3H-gld/gld.lpr/lpr (gld.lpr) and CBA-lprcg/lprcg (lprcg) mice were given a single i.p. dose of 30 mg/kg dimethylnitrosamine (DMN). Liver tissues were collected from mice killed 6, 12, 24 and 36 hrs post treatment, and the progression of the lesions was characterized morphologically and by the TUNEL method. DMN induced centrilobular hepatic injury accompanied with acute hemorrhage, and all mice died 36 to 48 hrs after the dosing. At 12 hrs after DMN administration, centrilobular hepatocytes revealed nuclear chromatin clumping. At 24 hrs, hepatocyte nuclei became fragmented to form apoptotic cells. Ultrastructurally, chromatin was condensed into a compact granular mass or crescent granular cap at the nuclear periphery. At 36 hrs, the number of apoptotic cells increased and they protruded into the sinusoid or were engulfed by the neighboring hepatocytes. A TUNEL-positive signal preceded the morphological changes and a few normal appearing centrilobular hepatocytes were positive 6 hrs post dosing. Endothelial damage was seen immunohistochemically at 24 hrs by disruption of type IV collagen and factor VIII-related antigen, resulting in massive hemorrhage in the centrilobular to mid zone. No inflammatory reactions were observed throughout the degeneration. The findings indicate that a single i.p. administration of DMN induced severe and fatal toxicity in liver tissues in mice which resembled human fulminant hepatitis. However, as gld-lpr and lprcg mice defective in apoptosis through the Fas system also showed similar severe liver damage, the Fas/Fas ligand system is not involved in DMN-induced liver apoptosis. No other organs or tissues were damaged, and the control mouse liver was intact.

An ultrastructural evaluation of acute 1-nitronaphthalene induced hepatic and pulmonary toxicity in the rat

1-Nitronaphthalene is a mutagenic particulate of diesel exhaust which causes acute liver and lung toxicity in rodents. The studies presented here describe morphological changes in the lung and liver at several time intervals following a single injection of 1-nitronaphthalene (100 mg/kg i.p.) in male Sprague-Dawley rats using transmission and scanning electron microscopy. Although both the lungs and liver are injured by 1-nitronaphthalene, the lungs appear to be the primary target organ. Within 4 h of treatment, all 1-nitronaphthalene treated animals exhibited respiratory distress characterized by labored breathing, severe gasping and chromodacryorrhea. The primary ultrastructural alteration were hydropic changes in the non-ciliated bronchiolar (Clara) cells of the distal-most bronchioles of the lung. These were apparent as early as 1 h after 1-nitronaphthalene injection, while adjacent ciliated cells showed no alterations. Over a 24 h period, the bronchioles showed progressive ultrastructural changes leading to necrosis and exfoliation of both ciliated and Clara cells. Interstitial pneumonitis and edema were observed in all animals treated with 1-nitronaphthalene, and was usually associated with bronchioles containing necrotic epithelium. In the liver, ultrastructural changes were observed in the centrilobular hepatocytes at 8 h and consisted of cytomegaly, loss of continuous inner membrane and reduced matrix density of the mitochondria. At 48 h, cellular damage to centrilobular hepatocytes was severe and nearly all mitochondria were damaged. Elevated levels of alanine aminotransferase, aspartate aminotransferase and bilirubin were detected in the serum of animals treated with 1-nitronaphthalene at 8–48 h. In conclusion, 1-nitronaphthalene is a pulmonary toxicant with a unique progression of injury, which primarily damages Clara cells followed by ciliated cells. This disparity is likely due to a difference in the bioactivation of 1-nitronaphthalene. Furthermore, this systemic toxicant also has injurious effects on the centrilobular region of the liver which precedes lung injury.

Coexpression of Ah Receptor and CYP1A1 in Hepatocytes of C57BL/6J and DBA/2J Mice

The distribution of the hepatic aryl hydrocarbon receptor (AhR) and CYP1A1 was investigated in control and 3-methylcholanthrene (3-MC)-treated C57BL/6J (B6) and DBA/2J (D2) mice. The AhR was identified as protein bands of 95 and 104 kDa in cytosol of B6 and D2 mice, respectively, while a protein of 93 kDa was detected in nuclear extracts from both murine strains. CYP1A1 was recognized as a protein of 56 kDa in microsomes from B6 and D2 mice. Lower amounts of immunodetectable AhR and CYP1A1 were observed in D2 mice, compared to B6 mice. Immunohistochemical studies were used to colocalize the AhR and CYP1A1 in adjacent liver sections. Staining for the AhR was localized most prominently in the cytoplasm of centrilobular hepatocytes and was minimal in the nuclei. Periportal hepatocytes were also reactive for the AhR, but the staining was usually localized in the nuclei. Other hepatocytes contained moderate amounts of cytoplasmic staining, whereas nuclear staining was present at low levels. Immunodetectable CYP1A1 also predominated in centrilobular hepatocytes and was negligible in periportal hepatocytes. The staining was exclusively cytoplasmic and was not seen in the nuclei of hepatocytes. Observations of adjacent liver sections revealed that the AhR and CYP1A1 were coexpressed in individual hepatocytes. A similar regional distribution for the AhR and CYP1A1 was manifested in B6 and D2 mice, with the exception that staining was more pronounced in the former under both control and induced conditions. These results showed heterogeneity in the distribution of the AhR and CYP1A1, with comparable levels of both proteins residing in the same hepatocytes. They further demonstrated that the AhR is localized in both the cytoplasm and nuclei of hepatocytes.

Lack of effect of piperonyl butoxide on unscheduled DNA synthesis in precision-cut human liver slices

In this study the effect of piperonyl butoxide (PBO) on unscheduled DNA synthesis in precision-cut human liver slices has been examined. Liver slices prepared from tissue samples from five human donors were cultured in medium containing [ 3H]thymidine and 0–2.5 mM PBO using a dynamic organ culture system. After 24 h the liver slices were processed for autoradiographic examination of UDS. As positive controls, liver slices were also cultured with three known genotoxic agents, namely 2-acetylaminofluorene (2-AAF), aflatoxin B 1 (AFB 1) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with >5 and >10 net grains. Compared to control liver slice cultures PBO had no effect on UDS. In contrast, treatment with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 and 0.02 mM AFB 1 and 0.005 and 0.05 mM PhIP produced significant increases in net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 mM PhIP. Treatment with 2-AAF, AFB 1 and PhIP also produced increases in the number of centrilobular hepatocyte nuclei with >5 and >10 net grains. At the concentrations examined neither PBO, 2-AAF nor PhIP had any significant effect on replicative DNA synthesis in 24 h cultured human liver slices. In cultured liver slices treated with 0.02, but not 0.002, mM AFB 1 a significant reduction in the rate of replicative DNA synthesis was observed. These results demonstrate that PBO does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice

Lobular distribution of human liver phenol and bilirubin uridine 5'- diphosphate glucuronosyltransferase messenger RNAs

BACKGROUND & AIMS: Heterogeneity in uridine 5'-diphosphate (UDP) glucuronosyltransferase expression across the human hepatic acinus may be important in the manifestation of certain zone-specific chemical hepatotoxicities. Previous immunohistochemical studies suggested that a phenol transferase induced by polycyclic aromatic hydrocarbons may be differentially expressed in centrilobular hepatocytes of rats. The aim of this study was to assess the distribution of the phenol and bilirubin transferases in human liver at the RNA level. METHODS: In situ RNA hybridization was used with two human liver samples and specific probes for the phenol transferase RNA, HLUG P1, and the bilirubin transferase RNAs, HUG-Br1 and HUG-Br2. RESULTS: The highest density signals were observed for the bilirubin transferase RNAs, both appearing to be evenly expressed in hepatocytes across the liver lobule. Slightly higher density of HUG-Br1 message was observed in some centrilobular hepatocytes surrounding larger central vein structures. HLUG P1 RNA was expressed at low levels (approximately fivefold greater than background signal) and was evenly distributed. CONCLUSIONS: The data suggest that a species difference exists in the distribution of the human and rat phenol transferase. No evidence was found for significant zonation in the pattern of expression of either the phenol or bilirubin transferase genes in human liver. (Gastroenterology 1996 Aug;111(2):472-80)

Overexpression of cytochrome P-450 isoforms involved in aflatoxin B1 bioactivation in human liver with cirrhosis and hepatitis.

Studies were carried out to test the hypothesis that inflammatory liver disease increases the expression of specific cytochrome P-450 isoenzymes involved in aflatoxin B1 (AFB) activation. The immunohistochemical expression and localization of various human cytochrome P-450 isoforms, including CYP2A6, CYP1A2, CYP3A4, and CYP2B1, were examined in normal human liver and liver with hepatitis and cirrhosis. The constitutive expression of CYP3A4 in normal liver showed a characteristic pattern of distribution in centrilobular hepatocytes, whereas CYP1A2, CYP2A6, and CYP2B1 were expressed uniformly throughout the liver acinus. In sections of liver infected with hepatitis B virus (HBV) or hepatitis C virus (HCV), the expression of CYP2A6 was markedly increased in hepatocytes immediately adjacent to areas of fibrosis and inflammation. CYP3A4 and CYP2B1 were induced to a lesser degree, and expression of CYP1A2 was unaffected. In HBV-infected liver, double immunostaining revealed that overexpression of CYP2A6 occurred in hepatocytes expressing the HBV core antigen. In HCV-infected liver, CYP2A6, CYP3A4, and CYP2B1 were overexpressed in hepatocytes with hemosiderin pigmentation. These results suggest that alterations in phenotypic expression of specific P-450 isoenzymes in hepatocytes associated with hepatic inflammation and cirrhosis might increase susceptibility to AFB genotoxicity.

Role of nutrition in the survival after hepatotoxic injury

Nutritional status is an important factor in determining susceptibility to toxic chemicals. While macro and micronutrients may affect many aspects of Stage I and Stage II of toxicity, in this paper, the influence of macronutrients as sources of energy required for cell division and tissue repair mechanisms on the outcome of hepatic injury is discussed. Male Sprague-Dawley rats maintained on normal rodent chow and 15% glucose (as a source of energy for the centrilobular hepatocytes) in drinking water for 7 days experienced an increased lethality from structurally and mechanistically different centrilobular hepatotoxicants (acetaminophen, thioacetamide, chloroform and carbon tetrachloride), while male Sprague-Dawley (S-D) rats fed rat chow containing palmitic acid (PA, 8% w/w, as a source of energy for the periportal hepatocytes) and l-carnitine (LC, 2 mg/ml, as a mitochondrial carrier for the supplemented fatty acids) in drinking water for 7 days were protected from a LD 100 dose (600 mg/kg, i.p.) of thioacetamide (TA). Indices of cell division revealed that cell cycle progression in the liver play a very critical role in determining the final outcome of hepatotoxic injury. These results confirmed our hypothesis that cell division and tissue repair play a critical role in survival after life-threatening hepatotoxic injury. Any manipulation directed towards altering a prompt and exacting compensatory cell division and tissue repair responses after hepatotoxic injury would also alter the final outcome of the toxicity. These studies indicate that the source of cellular energy can decisively influence the compensatory response of the target tissue to alter the outcome of hepatotoxic injury. Since nutritional status is known to vary widely among human populations, these could contribute enormously to susceptibility of human populations to toxic chemicals.

Structural and Functional Alterations of Hepatocytes during Transient Phalloidin-Induced Cholestasis in the Rat

To study the relationship between the dynamic actin web and bile secretion, we developed an acute model of cholestasis, using phalloidin, and examined sequential morphologic and biochemical events in rat liver. Biliary function (bile flow, bile, and canalicular membrane components) and cellular integrity (release of hepatic enzymes in serum and bile, canalicular structure, and microfilaments distribution) in rats given a single iv dose of phalloidin (0.8 mg/kg body weight) were assessed at 15, 45, and 90 min, 24 hr, and 5 days postinjection. Bile flow decreased significantly at 45 and 90 min, but cholestasis was transient since bile secretion returned to control levels at 24 hr. The biliary bile acid secretion rate was not modified during the same time period, indicating that cholestasis may have been due to impairment of the bile acid independent component of bile flow. Serum alanine aminotransferase and lactate dehydrogenase as well as biliary alkaline phosphatase and alkaline phosphodiesterase-1 activities were not altered by phalloidin treatment. These data, coupled with morphologic studies, provide no evidence of cell damage. Electron microscopy revealed that the pericanalicular actin web in both centrilobular and periportal hepatocytes was increased at 90 min and further enlarged at 24 hr and 5 days after phalloidin injection. At all time periods, the canalicular structure was well preserved. Na+K+–ATPase and Mg2+–ATPase activities in membrane fractions enriched in bile canalicular complexes decreased significantly at 15 min and remained low up to Day 5. Mg2+–ATPase activity returned to control levels by Day 5. The lipid constituents of liver cell membranes enriched in canalicular complexes showed no significant variations 90 min after toxin treatment but, at 24 hr, phospholipid content rose and membrane fluidity increased. These results clearly indicate that the bile flow variation after a single low dose of phalloidin can be dissociated from specific pericanalicular microfilament distribution, lending further support to the view that normal biliary function is not strictly dependent on the integrity of the actin filament network.

Expression of growth factors and growth factor receptors in the liver of C57BL/10J mice following administration of phenobarbitone.

Liver enlargement is a common feature of non-genotoxic rodent hepatocarcinogens administered at high doses. In the present study, the expression of growth factors and growth factor receptors was investigated in the C57BL/1OJ mouse during liver enlargement induced by the non-genotoxic rodent hepatocarcinogen, sodium phenobarbitone (PB). Male mice were dosed 0-2500 p.p.m. PB in the diet for 1, 4 and 13 weeks. There was a dose and time dependent increase in liver weight. Hepatocyte replication, assessed by incorporation of bromodeoxyuridine, was increased in a dose-dependent manner at week 1 only (18-fold increase at 2000 p.p.m.) and was predominantly localized in the centrilobular region. At week 1, PB (2500 p.p.m.) caused transient increases in transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR) and decreases in transforming growth factor beta1 (TGF-beta1) and mannose-6-phosphate receptor (M6PR) in centrilobular hepatocytes which correlated with the replication in this region. At week 1, there was an increase in both hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (HGFR) which colocalized in centrilobular hepatocytes; in some mice or periportal hepatocytes in other mice. After 13 weeks, HGF and HGFR were localized in the cytoplasm of centrilobular hepatocytes of all mice but exhibited a differential intracellular distribution across the lobule. At 2500 p.p.m. PB, EGFR and HGFR mRNA were essentially unchanged over the 13 week dosing period whilst M6PR mRNA was increased 2- to 4-fold. At 2500 p.p.m. PB, EGFR protein levels from immunoblots showed a consistent decrease over the 13 weeks whilst M6PR and HGFR protein levels were essentially unchanged. The protein level and mRNA data for EGFR suggest post-transcriptional modification. Thus, phenobarbitone caused transient replication of hepatocytes and modulation of growth stimulatory and inhibitory factors and their associated receptors in terms of overall levels and regional distribution in the liver.

Modulation of chemical-induced lung and liver toxicity by all- trans-retinol in the male Sprague-Dawley rat

We have previously shown that retinol pretreatment limits the amount of pulmonary injury caused by 1-nitronaphthalene in male Sprague-Dawley rats. The main objective of this study was to determine if retinol pretreatment can protect the lung from the toxicity of other systemic pneumotoxicants. Furthermore because retinol has been shown to alter the hepatotoxicity of several chemicals, a secondary objective was to evaluate its effects on the liver injury caused by these toxicants. Rats were pretreated with all- trans-retinol (75 mg/kg/day p.o.) for 1 week, and given 2-nitronaphthalene (200 mg/kg i.p.) or paraquat (25 mg/kg i.p.). At 24 h after 2-nitronaphthalene treatment, pulmonary morphological changes associated with the bronchiolar epithelium, as well as a moderate pneumonitis were observed. Pretreatment of rats with retinol inhibited the majority of 2-nitronaphthalene-induced pulmonary damage including the infiltration of inflammatory cells and associated edema. However, these animals possessed limited lesions associated with their non-ciliated bronchiolar epithelial (Clara) cells. Interestingly, pretreatment with retinol also caused a significant potentiation of 2-nitronaphthalene-induced liver damage. The potentiated hepatotoxicity consisted of centrilobular hepatocyte necrosis with infiltration of inflammatory cells. Gadolinium chloride (GdCl 3), an inhibitor of Kupffer cell function, significantly decreased the potentiated hepatocellular injury. In paraquat-treated rats focal areas of damage to the alveolar parenchyma, consisting of inflammatory cell infiltration and alveolar sac remodeling, were observed at 48 h. Pretreatment with retinol caused significant protection from the pulmonary damaged caused by paraquat. Specifically, there was a lack of alveolar parenchymal cell damage and inflammatory cell infiltration in these animals. From these experiments, we conclude that retinol pretreatment decreases the severity of 2-nitronaphthalene and paraquat-induced pulmonary toxicity, apparently by inhibiting the inflammatory responses associated with the progression of toxic injury. In the liver, retinol potentiated 2-nitronaphthalene-induced hepatotoxicity by a mechanism which directly involves Kupffer cells.

Hepatic lipid peroxidation in patients with hereditary hemochromatosis: [formula omitted] Departments of 1Clinical Chemistry and 2Anatomy, University of Oulu, Finland, 3Division of Gastroenterology and Hepatology, 4Department of Pathology, Saint Louis University School of Medicine, St. Louis, MO

Pathophysiological mechanisms for hepatocellular injury, fibrosis and/or cirrhosis in hepatic iron overload are poorly understood. An increase in intracellular transit pool of iron can catalyze peroxidation of lipids to produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Covalent binding of such lipid aldehydes with proteins may cause impairment in cellular function and integrity. This investigation was focused on quantitative determination of MDA and HNE-protein adducts, and to establish a correlation between iron deposition and formation and localization of MDA and HNE-protein adducts, using immunohistochemistry. To achieve iron overload, male SD rats were fed a 2.5% carbonyl iron-supplemented diet for six weeks, while control animals received standard diet. Total iron as well as low molecular weight chelatable iron (LMWC-Fe) in the hepatic tissue of rats fed the iron supplemented diet increased significantly (∼14- and ∼15-fold, respectively). Quantitative ELISA for MDA-and HNE-protein adducts showed remarkable increases of 186 and 149%, respectively, in the liver homogenates of rats fed the iron-supplemented diet. Sections of liver stained for iron showed striking iron deposits in periportal (zone 1) hepatocytes, which was less dramatic in midzonal (zone 2) cells. Livers from iron-loaded rats showed strong, diffuse staining for both MDA and HNE adducts, which was highly pronounced in centrilobular (zone 3) hepatocytes, but was also evident in midzonal cells (zone 2). The demonstration of greater formation of both MDA and HNE-protein adducts provides evidence of iron-catalyzed lipid peroxidation in vivo. Although in this model of iron overload there was no evidence of tissue injury, our results provide an account of some of the initiating factors or early molecular events in hepatocellular damage that may lead to the pathological manifestations seen in chronic iron overload.

ICAM-1 Expression in Hepatocytes Following Dissociation of Cell-to-Cell Contact in Rats

ICAM-1 was not detected immunohistologically in hepatocytes in normal rats, but detectable in centrilobular degenerative hepatocytes in carbon tetrachloride-intoxicated rats. ICAM-1 expression was observed even in normal hepatocytes following liver perfusion with Hank′s balanced salt solution at a flow rate of 4.2 mL/g liver weight/min or with the same solution containing collagenase or EGTA at the physiological flow rate (1.4 mL/g liver weight/min). Such expression was also observed when liver perfusion was performed after pretreatment of rats with cycloheximide. On electron microscopy, ICAM-1 was exclusively stained on hepatocyte plasma membrane that was detached from the plasma membrane of adjacent hepatocytes. ICAM-1 mRNA and ICAM-1 protein were detected in hepatocytes freshly isolated from normal rats. Thus, ICAM-1 expression in degenerative hepatocytes as well as in hepatocytes following liver perfusion can be assumed to result from dissociation of cell-to-cell contact.

Comparison of Styrene Hepatotoxicity in B6C3F1 and Swiss Mice

Comparison of Styrene Hepatotoxicity in B6C3F1 and Swiss Mice. Morgan, D. L., Mahler, J. F., Moorman, M. P., Wilson, R. E., Price, H. C., Jr., Richards, J. H., and O'Connor, R. W. (1995). Fundam. Appl. Toxicol. 27, 217-222. Inhalation exposure to styrene at concentrations that cause metabolic saturation results in significantly greater hepatotoxicity in B6C3F1 mice than in Swiss mice; females of both strains are more susceptible than males. These studies were conducted to investigate the mouse strain and gender differences in susceptibility to hepatotoxicity caused by repeated exposure to styrene at concentrations that do not cause metabolic saturation. Male and female B6C3F1 and Swiss mice (8 weeks old) were exposed to 0, 150, or 200 ppm styrene for 6 hr/day, 5 days/week, for up to 2 weeks. Changes in body and liver weights, serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) levels, liver histopathology, and total liver glutathione (GSH) were evaluated after 2, 3, 5, and 10 exposures (six mice/sex/strain/time point/concentration). Blood levels of styrene and styrene-7,8-oxide (SO) were measured in mice exposed to 200 ppm styrene for 2, 3, or 5 days (six mice/sex/strain/time point/concentration). Serum ALT and SDH levels were significantly elevated only in female B6C3F1 mice after 3 exposures to 200 ppm styrene; enzyme levels had returned to control levels when measured after 5 and 10 exposures. Degeneration and coagulative necrosis of centrilobular hepatocytes were observed in female B6C3F1 mice exposed 2, 3, and 5 days to 150 or 200 ppm styrene; incidences of these lesions were greater in the 200 ppm than in the 150 ppm dose group. After 10 days of exposure to 150 or 200 ppm styrene, hepatocellular lesions had resolved, although a residual chronic inflammation was present in livers of most female B6C3F1 mice. Degeneration of centrilobular hepatocytes was observed in one male B6C3F1 mouse after 3 exposures to 200 ppm, and no significant lesions were observed in livers of exposed Swiss mice. Significant dose-related decreases in hepatic GSH were observed in both sexes of both strains throughout the 2-week exposure. In general, hepatic GSH depletion was greatest in female B6C3F1 mice. Exposure to 200 ppm caused 60-70% GSH depletion in female B6C3F1 mice at each time point. GSH depletion generally decreased in B6C3F1 mice and increased in Swiss mice with continued exposure to 150 ppm styrene. With continued exposure to 200 ppm, GSH depletion generally decreased in all mice. Blood styrene and SO levels increased in all groups with the number of exposures. Styrene levels were significantly higher in B6C3F1 mice than in Swiss mice; however, within each strain gender differences were not significant. These data suggest that the transient hepatotoxicity in female B6C3F1 mice was related to greater hepatic GSH depletion and/or slower GSH regeneration in these animals.

A multidisciplinary approach to toxicological screening: I. Systemic toxicity.

The toxicity of 10 chemicals, including pesticides (carbaryl, chlordane, heptachlor, and triadimefon), solvents (carbon tetrachloride, dichloromethane, tetrachloroethylene, and trichloroethylene), and industrial chemicals [diethylhexylphthalate (DEHP) and phenol] was examined in the liver, kidneys, spleen, thymus, and adrenals of female F344 rats after 1 or 14 d of oral dosing. For each chemical, 4 doses were based on fractions of the acute LD50, which was estimated using an abbreviated (up-and-down) method. A multivariate analysis (MANOVA) was conducted for each organ using selected measures of toxicity. A post hoc contrast analysis was also conducted for significant MANOVA results in order to determine effective and ineffective doses. A single dose of heptachlor resulted in necrotic lymphocytes in the spleen and thymus at doses > or = 23 mg/kg. Triadimefon altered liver and spleen weights; this effect has not been described previously. Dichloromethane (> or = 337 mg/kg/d for 14 d) increased the incidence of necrosis of individual centrilobular hepatocytes. Trichloroethylene-induced hepatotoxicity was obtained at doses an order of magnitude lower than those reported in the literature. Acute DEHP (150 mg/kg) increased mitotic figures in hepatocytes, which were replaced by hepatocellular cytomegaly after 14 d of dosing at the same level. Following phenol exposure, there was an increased incidence in hepatocellular necrosis at 1 d, and an increased incidence of kidney lesions at 1 and 14 d; these findings were considered to be the result of vascular stasis. Overall, the algorithm used to select doses was effective for both 1- or 14-d dosing regimens. For all chemicals except carbon tetrachloride, the lowest effective dose for systemic toxicity was within the range of 3-56% of the LD50 for acute dosing, and 1-30% of the LD50 for repeated administration.

Two-generation reproduction toxicity study with isopropanol in rats.

A two-generation reproduction toxicity study was conducted in rats with isopropanol. Thirty rats of each sex per group (P1) were dosed once daily by oral gavage with 0, 100, 500 or 1000 mg isopropanol kg-1 for at least 10 weeks prior to mating. Parental animals were mated within groups for up to 3 weeks. Parental females were dosed during mating, gestation and lactation; parental males were dosed during mating through delivery of their last litter sired. The P2 adults were selected from the F1 litters and were dosed for 10-13 weeks before mating to produce a single litter. Findings in the parental animals included increased lactation body weight gain in the mid- and high-dose females, increased liver and kidney weights in the mid- and high-dose groups of both sexes and centrilobular hepatocyte hypertrophy in some P2 males. There was also accumulation of hyaline droplets and other microscopic findings in the kidneys from the mid- and high-dose P1 males and from all treated groups of the P2 males. Increased mortality was observed in the high-dose F1 offspring during the early postnatal period, although no other clinical signs of toxicity were observed in the offspring of either generation. In addition, offspring body weight was reduced during the early postnatal period in the high-dose F1 males and in the high-dose F2 pups of both sexes. Eighteen out of 70 F1 weanlings in the 1000 mg kg-1 group died or were euthanized prior to P2 selection. No treatment-related post-mortem findings were observed in the offspring from either generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical Localization of Acetaminophen in Target Tissues of the CD-1 Mouse: Correspondence of Covalent Binding with Toxicity

Immunohistochemical Localization of Acetaminophen in Target Tissues of the CD-1 Mouse: Correspondence of Covalent Binding with Toxicity. Emeigh Hart, S. G., Cartun, R. W., Wyand, D. S., Khairallah, E. A., and Cohen, S. D. (1995). Fundam. Appl. Toxicol. 24, 260-274.Administration of hepatotoxic doses of acetaminophen (APAP) to mice results in necrosis, not only of liver cells but of renal proximal tubules and bronchiolar and olfactory epithelium. In the liver, covalent binding is localized to the centrilobular hepatocytes which later undergo necrosis. This study was undertaken to compare the cellular distribution of bound APAP in all four major target tissues with that of cytochrome P4502E1 (a P450 isoenzyme commonly associated with APAP bioactivation), with emphasis on the cell types which later undergo necrosis. Tissues were collected from mice at selected times after APAP administration (600 mg/kg, po) and fixed by microwave irradiation for immunohistochemistry, or in formalin for histopathological study. Immunohistochemical localization of bound APAP was performed on 5 μm paraffin sections using an affinity-purified anti-APAP antibody. Similar tissues from naive mice were used for immunohistochemical localization of cytochrome P4502E1 (using a polyclonal sheep anti-P4502E1 antibody). Positive staining with both the anti-APAP and the anti-P4502E1 antibodies was similar in distribution, being present in the cell types which become damaged by APAP in all four target tissues. These results demonstrate that covalent binding and subsequent necrosis are localized in common with cytochrome P4502E1, suggesting that, as in the liver, toxicity in extrahepatic targets is also related to the ability of these tissues to activate APAP in situ.

Induced regenerative cell proliferation in livers and kidneys of male F-344 rats given chloroform in corn oil by gavage or ad libitum in drinking water

These studies were designed to establish the dose response relationships for the induction of cytolethality and regenerative cell proliferation in the liver and kidneys of male F-344 rats given chloroform by gavage or in drinking water. Rats were administered oral doses of 0, 10, 34, 90 or 180 mg/kg/day chloroform dissolved in corn oil by gavage for 4 days or for 5 days/week for 3 weeks. A second group of rats was given chloroform ad libitum in the drinking water at concentrations of 0, 60, 200, 400, 900 or 1800 ppm for 4 days or 3 weeks. Bromodeoxyuridine (BrdU) was administered via an implanted osmotic pump 3.5 days prior to necropsy to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the labelling index (LI) evaluated as the percentage of S-phase cells. Rats treated with 90 or 180 mg/kg/day by gavage for 4 days had mild to moderate degeneration of renal proximal tubules and centrilobular hepatocytes. These alterations were absent or slight after 3 weeks of treatment. LI were increased in the kidney cortex only in the rats treated with 180 mg/kg/day for 4 days. A dosedependent increase in LI was seen in rat liver after 4 days of treatment with 90 and 180 mg/kg/day by gavage, but the LI remained elevated after 3 weeks of treatment only at the 180 mg/kg/day dose. When chloroform was administered in the drinking water, no microscopic alterations were seen in the kidneys after 4 days of treatment. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were noted in the controls, but no clear dose response relationship was evident. However, the overall renal LI was not increased at any dose or time point. Similarly, only mild hepatocyte vacuolation was observed in rats given 1800 ppm chloroform in the water for 3 weeks with no increase in the hepatic LI at any time point, even though the rats were consuming chloroform at a rate of 106 mg/kg/day at the 1800 ppm drinking water concentration. These data indicate more severe hepatic and renal toxicity when chloroform is administered by gavage than in the drinking water and a different pattern of regenerative proliferation in the kidney. Thus, the impact that different methods and rates of chloroform administration have on tissue dosimetry and tissue response must be understood in interpreting toxic outcomes in rodents and extrapolating potential effects to humans.

Immunohistochemical Localization of Acetaminophen in Target Tissues of the CD-i Mouse: Correspondence of Covalent Binding with Toxicity

Administration of hepatotoxic doses of acetaminophen (APAP) to mice results in necrosis, not only of liver cells but of renal proximal tubules and bronchiolar and olfactory epithehum. In the liver, covalent binding is localized to the centrilobular hepatocytes which later undergo necrosis. This study was undertaken to compare the cellular distribution of bound APAP in all four major target tissues with that of cytochrome P4502E1 (a P450 isoenzyme commonly associated with APAP bioactivation), with emphasis on the cell types which later undergo necrosis. Tissues were collected from mice at selected times after APAP administration (600 mg/kg, po) and fixed by microwave irradiation for immunohistochemistry, or in formalin for histopathological study. Immunohistochemical localization of bound APAP was performed on 5-μm paraffin sections using an affinity-purified anti-APAP antibody. Similar tissues from naive mice were used for immunohistochemical localization of cytochrome P4502E1 (using a polyclonal sheep anti-P4502E1 antibody). Positive staining with both the anti-APAP and the anti-P4502E1 antibodies was similar in distribution, being present in the cell types which become damaged by APAP in all four target tissues. These results demonstrate that covalent binding and subsequent necrosis are localized in common with cytochrome P4502E1, suggesting that, as in the liver, toxicity in extrahepatic targets is also related to the ability of these tissues to activate APAP in situ.

The Localization of HCV and the Expression of Fas Antigen in the Liver of HCV-related Chronic Liver Disease.

We have investigated the localization of HCV in the liver and the expression of Fas antigen, involved in hepatocellular death through apoptosis, to examine the relation between HCV infection and Fas antigen cellular expression. The localization of HCV was investigated by in situ hybridization (ISH), and the expression of Fas antigen was determined by immunohistochemistry using an anti-Fas monoclonal antibody, in 20 surgically-resected liver specimens from anti-HCV positive patients. The Methylgreen/Pyronin Y technique clearly showed well-preserved tissue RNA in 8 of 20 livers. ISH determined the co-localization of signals for plus and minus strands of HCV-RNA in the cytoplasm of hepatocytes. Fas antigen was more strongly expressed on the plasma membrane of the periportal hepatocytes than on the plasma membrane of the centrilobular hepatocytes, even when HCV-RNA was found to be widely distributed in the hepatocytes throughout the liver lobule. Furthermore, Fas antigen positive hepatocytes were frequently observed close to the area of piecemeal necrosis associated with a high incidence of apoptosis. The results suggested that the expression of Fas antigen does not occur by HCV infection alone, but probably requires several other factors.

Rapid appearance of connexin 26-positive gap junctions in centrilobular hepatocytes without induction of mRNA and protein synthesis in isolated perfused liver of female rat

In the adult rat liver, the gap junction protein connexin 32 (Cx32) is evenly distributed in hepatocytes within the liver lobules, while connexin 26 (Cx26) is preferentially localized in hepatocytes in periportal zones. We report here that Cx26-positive gap junctions rapidly appear in the centrilobular hepatocytes of adult female rat livers during a 30 minute perfusion of the liver through the hepatic portal vein with a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and oxygen transport FC-43 fluid at a physiological flow rate without any changes in the distribution of Cx32. The change in the localization of Cx26 was closely related to that of E-cadherin, and there was no significant increase in the amounts of Cx26 protein and mRNA. The appearance of Cx26 in the centrilobular hepatocytes was inhibited by treatment with cytoskeleton disruptors such as colchicine and cytochalasin B, and intracytoplasmic transport inhibitors such as brefeldin A. The liver perfusion induced the appearance of Cx26 in the centrilobular hepatocytes only in female rats. Estrogen treatment of ovariectomized rats caused the appearance of both Cx26 and E-cadherin in centrilobular hepatocytes not only in the perfused liver but also in the non-perfused liver. Our results indicate that in the rat liver: (a) the localization of Cx26 can be modulated by a post-translational mechanism; (b) E-cadherin may play an important role in the formation of gap junctions composed of Cx26; and (c) the formation of gap junctions is regulated by female steroid hormones.