A histopathological and stereological study of liver damage in female rats caused by mercury vapor

We examined the possible effects of elemental mercury vapor on the liver of the female rats. We divided the animals into an untreated control group and an experimental group that was exposed to mercury vapor for 45 days. Liver samples were obtained for histological and stereological analysis. The total liver, parenchyma and sinusoid volumes were increased significantly in the mercury vapor treated group compared to controls. Also, the mean density, total number and mean nuclear diameter of hepatocytes, except for binucleated hepatocytes, was decreased in the experimental group compared to controls. Light and electron microscopy revealed alterations of liver structure of the experimental animals compared to controls.     

Effect of ischemia-reperfusion on the heterogeneous lobular distribution pattern of glycogen content and glucose-6-phosphatase activity in human liver allograft.

In order to examine glucose metabolism in liver grafts after cold ischemia and reperfusion, the heterogeneous lobular distribution pattern of glycogen content and glucose-6-phosphatase activity was studied using histochemical methods. The characteristic heterogeneous lobular distribution pattern of glycogen and glucose-6-phosphatase was maintained after preservation and reperfusion. However, it appeared that glycogen content decreased in both periportal and centrilobular hepatocytes after reperfusion. The glycogen decrease was higher in periportal hepatocytes. Glucose-6-phosphatase activity was maintained after reperfusion in most of the cases in periportal hepatocytes. In centrilobular hepatocytes, more cases showed a decrease in enzyme activity. It is suggested that ischemia-reperfusion mainly affects the glycogen content in both periportal and centrilobular hepatocytes and that centrilobular glucose-6-phosphatase activity is more sensitive to ischemia-reperfusion injury than periportal hepatocytes.     

Content and transportation of Ca(2+) to hepatocytes and lipid content of liver cell membrane structure in experimental diabetes in the rat

It is proved that at experimental diabetes the calcium content in hepatocytes is disturbed. This disorder is mostly shown in increase of calcium content in hepatocytes and in decrease of accumulation of these ions in mitochondrias. One of the possible reasons of changes at this pathology is the change of lipid content of hepatocytes, mitochondrias and microsomes. It is proved that the in vitro model systems Ca ions inhibits the synthesis of 25-hydroxyvitamin D3 by hepatocytes.     

Peri- and postnatal changes in reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase content in hepatocytes of rats

Summary To study the process of the expression of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase (EC 1.6.2.4) in the liver during development, the amount of enzyme in the cytoplasm of periportal and perivenular hepatocytes in sections cut from livers of male rats was measured during peri- and postnatal growth by quantitative immunohistochemistry with a video image processor. In livers of 19-day-old foetuses, the reductase content in the cytoplasm of periportal and perivenular hepatocytes was 0.16 μM and 0.20 μM, respectively. From the 19th day of gestation to 5 days after birth, the enzyme content increased markedly in the cytoplasm of periportal (288%) and perivenular hepatocytes (301%). Subsequently, the content in the cytoplasm of periportal hepatocytes increased slightly (46%) from 5 to 20 days of age, remained unchanged from 20 to 45 days of age, and increased slightly (15%) from 45 to 90 days of age. However, the content in the cytoplasm of perivenular hepatocytes increased progressively (125%) between 5 and 90 days of age. Thus, the amount of cytochrome P-450 reductase increases markedly in periportal and perivenular hepatocytes during the perinatal period, and subsequently the enzyme content increases gradually in periportal hepatocytes and progressively in perivenular hepatocytes. The present results also suggest that the divergence between cytochrome P-450 expression and the cytochrome P-450-dependent drug metabolic activity in hepatocytes during the perinatal period, found in previous studies, can be attributed to a low cytochrome P-450 reductase density in the membrane of endoplasmic reticulum of periportal and perivenular hepatocytes.     

The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments

Using absorption and fluorescent cytophotometry methods, glycogen contents were studied in hepatocytes located in liver lobules and in hepatocytes, which make the general population of these cells in normal and cirrhotic rat liver. In cirrhosis, the content of glycogen in hepatocytes located in lobules obviously rises in comparison with the norm, but to a lesser degree, than in hepatocytes making the general population of these cells in cirrhotic liver. The content of glycogen in hepatocytes, located in lobules of pathologically changed liver in bemithyl treated rats, did not differ from the norm. At the same time, the glycogen content in hepatocytes, representing the general population of these cells in cirrhotically altered bemithyl injected rat liver, remained higher than in the norm. The data obtained indicate that distinctions in particular cell microinvironment, obviously present in cirrhotic liver, render essential influence on hepatocyte functional activity.     

Measurement of NADPH-ferrihemoprotein reductase content in sections of liver

We developed a method for measuring the content of NADPH-ferrihemoprotein reductase in sections of liver. First, reductase in sections of rat liver was detected with the indirect immunoperoxidase reaction. Subsequently, specific absorbances were measured in the stained sections by microphotometry. Then, the resulting specific absorbances were converted into the reductase content in the sections using an apparent extinction coefficient obtained from a nitrocellulose binding assay. The average of the reductase content in hepatocytes in periportal, intermediate, and perivenous zones thus measured was consistent with the value in liver homogenates estimated by enzyme-linked immunosorbent assay. Therefore, the present method gave accurate measurement of the reductase content in the sections. Perivenous hepatocytes contained 1.5 times as much reductase (1.15 nmol/g liver, mean for five animals) as that in periportal hepatocytes (0.74 nmol/g liver). The reductase content in hepatocytes in the intermediate zone (0.93 nmol/g liver) was intermediate between values of the periportal and perivenous hepatocytes.     

Effects of EGF on the mass of inositol 1,4,5-trisphosphate and SN(1,2)-diacylglycerol in freshly isolated rat hepatocytes: comparison with vasopressin.

We measured the masses of inositol 1,4,5 trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG) in hepatocytes in response to both epidermal growth factor (EGF) and vasopressin. EGF at 25 nM did not alter Ins(1,4,5)P3 content of hepatocytes. However, the combination of 100 nM EGF concentration and incubation with lithium did increase Ins(1,4,5)P3 content. This increase was only one tenth of that elicited by vasopressin in parallel incubations. This finding resolves a controversy concerning the ability of EGF to increase Ins(1,4,5)P3 in hepatocytes, and argues against a role for phosphoinositide hydrolysis in EGF action in hepatocytes. Both EGF and vasopressin caused a rapid (30 s) increase in DAG content. A delayed increase in DAG content, that was maximal after several minutes, was observed only for vasopressin. The rapid increase in DAG content implies an activation of protein kinase C for both EGF and vasopressin.     

Effect of prior nutritional status on the activity of lipogenic enzymes in primary monolayer cultures of rat hepatocytes.

Effect of prior nutritional status of the animal on the activity of lipogenic enzymes and the fatty acid content of cultured hepatocytes was investigated. Hepatocytes were isolated from rats that were starved for 24 h ('starved') or continuously fed ('fed'), or starved for 48 h and then re-fed for 48 h ('re-fed') with a carbohydrate-rich fat-free diet, and maintained as monolayer cultures for 96 h in a serum-free glucose-rich medium (Waymouth's MB752/1) supplemented with insulin, dexamethasone and tri-iodothyronine. The fatty acid content and the activities of acetyl-CoA carboxylase, fatty acid synthase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were determined initially at 3 h after plating and then every 24 h. Initially the activities of all the four enzymes were highest in hepatocytes isolated from the re-fed rats and lowest in those from the starved rats. With time in culture, the activity of all these enzymes increased severalfold (2-5, depending on the enzyme under consideration) in hepatocytes isolated from fed and starved rats, whereas there was a severalfold (2-5) decrease in the activity of these enzymes in hepatocytes isolated from re-fed rats. The initial fatty acid content of the hepatocytes from re-fed rats was 2-3 times that in the other two groups of hepatocytes. The fatty acid content seemed to increase in all three groups of hepatocytes during the 96 h in culture, but these apparent increases were not statistically significant.     

Isoproterenol-induced Mg2+ uptake in liver.

Isoproterenol increased the Mg2+ content of hepatocytes after injection into rats or after addition to collagenase-dispersed hepatocytes. cAMP also the increased cellular Mg2+ content of isolated hepatocytes. This effect was prevented by staurosporine. Phorbol ester had no effect on the Mg2+ content of isolated hepatocytes, and after injection of isoproterenol into rats, protein kinase C of liver was not affected. It was concluded that isoproterenol induced long-term Mg2+ influx via the activation of protein kinase A which can be inhibited by staurosporine.     

Biochemical study of cultured hepatocytes obtained from normal adult rats]

A method has been developed for the preparation of hepatocytes that contained 98.6% of that cell type with a yield of 50%. No change was detected in the DNA content per nucleus but losses were noted in RNA and proteins in freshly isolated hepatocytes. The hepatocytes have been kept alive for long periods in suspension or static cultures without any mitoses being observed. However DNA content doubled over a period of two weeks. This level was maintained during the third week. Tritiated thymidine incorportation similarly increased for a period of two weeks and then remained constant. RNA content went down rapidly to one fourth of the original level and remained unchanged thereafter. The protein content did not vary significantly. Incorporation of labelled leucine into proteins has also been measured at various periods of time following isolation of the cells. Even after 62 days in suspension culture, the hepatocytes continued to incorporate leucine into proteins. The incorporation of leucine proceeded through the normal pathway since the presence of t-RNA-bond-leucine was observed. The results reported here suggest that the hepatocytes are arrested in the G2 phase of the cell cycle.     

The possible connection of adenylic nucleotides to the rhythm of protein synthesis in hepatocytes in vitro

Circahoral protein synthesis rhythms observed in hepatocytes in vitro differ in phase from the oscillations of intracellular amount of ATP. ATP added to culture medium interferes with the rhythms of protein synthesis and intracellular DNA content. Addition of ADP increases both the incorporation level of hepatocytes and ATP content. The data obtained has been discussed.     

Glutathione synthesis during the rewarming of rat hepatocytes preserved in the University of Wisconsin solution

In this study, we used isolated rat hepatocytes to investigate the effect of nucleoside content of the preserved cells on the ability to synthesize glutathione (GSH) during the rewarming process. We cold-stored hepatocytes in University of Wisconsin (UW) solution (72 h, 0 degrees C, N(2)) without nucleosides and with the addition of 5 mM adenosine or 10 mM ATP. After 72 h of cold storage, we determined the GSH synthesis rate and the ATP content of the cells. We found a GSH synthesis rate similar to that of freshly isolated hepatocytes only in the group of cells cold-stored with 10 mM ATP. When we tested the cellular ATP concentrations, we found that controls and preserved cells with 10 mM ATP showed a similar value of ATP during the rewarming step. Our results suggested that the incorporation of ATP in the UW solution increased the ATP content and the rate of GSH synthesis of cold-stored hepatocytes during rewarming.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

Glycogen distribution in rat hepatocyte populations after a single exposure to 4-dimethylaminoazobenzine and subsequent injections of phenobarbital

7 day after a single interperitoneal injection of carcinogen 4-dimethylaminoazobenzen (DAB), a little number of cells with high glycogen contents was found in parallel with a decreased glycogen content in most isolated hepatocytes. 1.5 months after DAB injection, the normal distribution of glycogen content was seen restored in hepatocytes. The treatment of rats with phenobarbital (6 PhB injections 7 days after DAB application) blocked the restoration of the normal glycogen distribution. 2 months after the last PhB injection (3 months after DAB injection) an increased glycogen content was found in the smallest hepatocytes.     

Influence of Obstructive Cholestasis and Sex Hormones on the Ratio of mRNA of Two Alternative Prolactin Receptor Isoforms in Rat Hepatocytes

The effects of obstructive cholestasis and sex hormones on the total content of prolactin receptor mRNA and ratio of mRNAs of its short and long isoforms have been studied in rat hepatocytes. Obstructive cholestasis caused insignificant changes in total content of prolactin receptor mRNA, but the proportion of mRNA of the long isoform increased. Comparison of prolactin receptor mRNA levels in gonadectomized and intact animals revealed opposite effects of male and female sex hormones on total mRNA, but both groups of these hormones increased the proportion of prolactin receptor short form mRNA. Changes in ratio of mRNA of receptor isoforms found in rat hepatocytes under obstructive cholestasis did not depend on levels of sex hormones. Obstructive cholestasis and sex hormones are suggested to regulate the content of long and short prolactin receptor isoforms in hepatocytes independently.     

Increased affinity to concanavalin A and enhanced secretion of alpha 1-acid glycoprotein by hepatocytes isolated from turpentine-treated rats

Hepatocytes were isolated from adult rats at various times after subcutaneous injection of turpentine (1 ml). The affinity to concanavalin A (Con A) of alpha 1-acid glycoprotein (AGP) and the intracellular content and rate of secretion of AGP and albumin were evaluated over a period of 19 days. Inflamed hepatocytes secreted mainly the Con A-reactive form of AGP whereas control hepatocytes secreted a higher amount of the Con A-non-reactive form. The intracellular content and rate of secretion of AGP by inflamed hepatocytes increased markedly whereas those of albumin decreased. However, when the residence time (ratio of intracellular content to rate of secretion) was evaluated, it appeared that the efficiency of secretion of both proteins was higher than in control hepatocytes. The changes in the affinity of AGP to Con A and in the secretion of AGP and albumin were reversible. These findings indicate that acute inflammation leads to posttranslational alterations during the intracellular transit of these secretory proteins.     

Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from experimental-diabetic rats

Isolated hepatocytes from rats with experimental diabetes exhibit increased content of cytochrome P-450 and cyclic AMP and normal activities of the regulatory enzymes delta-aminolevulinic acid synthase and ferrochelatase. The inducing effect exerted by phenobarbital on cytochrome P-450, delta-aminolevulinic acid synthase and ferrochelatase biosynthesis and cyclic AMP content in diabetic hepatic cells is markedly greater than that observed in normal hepatocytes. This stimulatory response is neither enhanced by added dibutyryl cyclic AMP nor repressed by glucose. The present results suggest that the heme pathway of diabetic hepatocytes is more susceptible to porphyrinogenic factors.     

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatocytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptor-defective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis.     

Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: influence on cellular DNA and protein content monitored by flow cytometry

Primary hepatocytes were cultured at oxygen tensions similar to those reported to be present in periportal (13% O2) and pericentral (4% O2) regions of the liver lobules. Cellular DNA and protein content of individual hepatocytes were determined simultaneously by two-parameter (DNA/protein) flow cytometry after 1, 4, and 7 days in culture. pO2 tensions monitored on line in conventional plastic culture dishes revealed that the depletion of the pO2 in the culture medium depended on the number of hepatocytes plated. When cultured as monolayer after 4-7 days at periportal (13% O2) and more pronounced at pericentral oxygen concentration (4% O2), up to 90% of the hepatocytes showed degenerated nuclei but normal protein content. By using culture dishes with teflon membrane bottoms the oxygen tension in the culture medium was accurately maintained by the incubator atmosphere. At pericentral oxygen tension the fraction of 2N cells increased by about 20%. That of the 4N cell was not affected, and the contribution of 8N hepatocytes dropped to 70% compared to cultures at periportal oxygen tension. Concomitantly, in the 4% O2 hepatocyte cultures the protein content of the 2N and the 4N cells was better preserved and increased by up to 10%. These results suggest that in vitro at pericentral oxygen conditions (4% O2) ageing of hepatocytes is delayed, regenerating processes are better maintained, and, furthermore, freshly isolated 4N hepatocytes have the potency to adapt their metabolism in vitro to periportal as well as to perivenous oxygen tensions.     

Differential in vitro hepatotoxicity of troglitazone and rosiglitazone among cryopreserved human hepatocytes from 37 donors

We report here our studies on troglitazone and rosiglitazone cytotoxicity in human hepatocytes isolated from multiple donors to investigate factors responsible for individual differences in sensitivity to the known hepatotoxicity of these antidiabetic drugs. Using cellular adenosine triphosphate (ATP) content as an endpoint, cytotoxicity of both drugs was evaluated in cryopreserved human hepatocytes from 37 donors. We confirmed reports of others that troglitazone was cytotoxic to human hepatocytes using cellular ATP content as an endpoint. In addition, we found that rosiglitazone, although less toxic in the study population, was cytotoxic to hepatocytes in some donors (EC(50)<100 microM). ATP content, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) metabolism, depletion of intracellular glutathione, Alamar Blue metabolism, and neutral red uptake were used as endpoints in a single donor study using freshly isolated human hepatocytes. Troglitazone appeared to be more toxic than rosiglitazone by all endpoints. From the demographic data provided to us for each donor, we were able to establish no direct correlation between cytotoxicity (expressed as EC(50) values) and age, sex, smoking status, or alcohol consumption. We conclude that troglitazone and rosiglitazone are differentially toxic to human hepatocytes, and that toxicity may be independent of age, sex, tobacco use, and alcohol use.