Ritva P. Evarts

Cellular distribution of transforming growth factor-beta 1 and procollagen types I, III, and IV transcripts in carbon tetrachloride-induced rat liver fibrosis.

The cellular distribution and temporal expression of transcripts from transforming growth factor-beta 1 (TGF-beta 1) and procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) genes were studied in carbon tetrachloride (CCl4)-induced rat liver fibrosis by using in situ hybridization technique. During the fibrotic process, TGF-beta 1 and procollagen genes were similarly and predominantly expressed in Desmin-positive perisinusoidal cells (e.g., fat-storing cells and myofibroblasts) and fibroblasts and their expression continued to be higher than those observed in control rats. These transcripts were also observed in inflammatory cells mainly granulocytes and macrophage-like cells at the early stages of liver fibrosis. The production of extracellular matrix along small blood vessels and fibrous septa coincided with the expression of these genes. Expression of TGF-beta 1 and procollagen genes were not detected in hepatocytes throughout the experiment. No significant differences in cellular distribution or time course of gene expression among procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) were observed. Desmin-positive perisinusoidal cells and fibroblasts appeared to play the principal role in synthesis of collagens in CCl4-induced hepatic fibrosis. The simultaneous expression of TGF-beta 1 and procollagen genes in mesenchymal cells, including Desmin-positive perisinusoidal cells, during hepatic fibrosis suggests the possibility that TGF-beta 1 may have an important role in the production of fibrosis.

Hepatic Stem Cell Compartment: Activation and Lineage Commitment

There is increasingly robust experimental evidence in support of the presence of a pluripotent cell compartment in the liver 1 , 2 , 3 , 4 , 5 , 6 , 7 . This compartment can under certain conditions function as a stem cell compartment and provide the needed progeny for regeneration of the hepatic parenchyma 8 , 9 . In the adult rat, specific conditions can be utilized to induce proliferation of a distinct population of small epithelial cells in the ductal structures of the liver 10 , 11 . These cells, conventionally described as oval cells, are characterized by ovoid nuclei and basophilic cytoplasma 10 , and display features of both bile duct cells and fetal hepatocytes 11 , 12 , 13 . There are three experimental systems, two in the rat and one in the mouse, in which it has been conclusively demonstrated that oval cells are capable of differentiation into hepatocytes 8 , 11 , 14 . The developmental potential of oval cells is, however, not restricted to hepatic lineages. Oval cells can differentiate into intestinal-type epithelia, and have been implicated in the development of pancreatic tissues 8 , 11 , 15 , 16 , 17 ; Fig. 1). The observations that sub-populations of proliferating oval cells phenotypically similar to early hepatoblasts, and that oval cells originate in or around the ductular structures in the portal area, strongly support the notion that the hepatic stem cell compartment resides in these structures 2 , 7 , 9 . Furthermore, present evidence clearly indicates that the hepatic stem cell compartment functions as a facultative stem cell compartment that is activated when the parenchymal cells are unable to proliferate in response to growth stimuli 2 , 8 , 18 , 19 .

Induction of microsomal dimethylnitrosamine demethylase by pyrazole

Pyrazole, a potent inhibitor of alcohol dehydrogenase, was found to be a potent inducer of the activity of low Km dimethylnitrosamine demethylase (DMN-d). One injection of pyrazole (200 mg/kg body wt) to weanling Wistar rats changed the microsomal DMN demethylase activity by 1.7, 1.9 and 2.5 times the control values at 6, 12 and 24 hr after the injection respectively. Pyrazole administration reduced arylhydrocarbon hydroxylase (AHH) activity. When animals were injected with pyrazole (200 mg/kg body wt) for 1, 2, 3 or 4 consecutive days, the values for DMN-d activity were 277, 297, 306 and 319% of the control values. The corresponding values for AHH were 91, 67, 57 and 45% for 1, 2, 3 and 4 injections respectively. pyrazole-induced DMN-d activity was NADPH dependent and was inhibited by CO; n-butanol gave a 50% inhibition at a concentration of 2 X 10(-3) M. The corresponding value for metyrapone was 1 X 10(-2) M. Cytochrome P-450 was slightly increased by pyrazole and its CO-complex gave an absorption maximum around 451 nm. When the microsomal proteins were separated using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, a large increase in a band at about 51,000 daltons was found in the liver microsomes of pyrazole-treated animals.

Effects of indole and tryptophan on cytochrome P-450, dimethylnitrosamine demethylase, and arylhydrocarbon hydroxylase activities

Abstract The effects of indole and l -tryptophan feeding on the activities of two liver microsomal enzymes, dimethylnitrosamine demethylase (DMN-d) and arylhydrocarbon hydroxylase (AHH), were studied in rats and hamsters. Both indole and tryptophan increased cytochrome P-450 concentration and DMN-d activity in rats, but only indole was effective in hamsters. AHH activity in both species was induced by indole. Both indole and tryptophan increased the ratio A 392–500nm / A 410–500nm obtained by n -octylamine difference spectroscopy of rat liver microsomes but not of hamster liver microsomes. Indole increased the absorbance difference ratio A 455–500nm / A 430–500nm of rat liver cytochrome P-450 obtained by ethyl isocyanide. The opposite was true for hamsters, which showed a low band at 455 nm. Indole gave a type II binding spectrum with rat liver microsomes, but the values for absorption minimum and maximum for tryptophan were somewhat outside the reported values for type II binders. Indole which induced liver AHH activity, protected against 7,12-dimethylbenz[a]anthracene (DMBA)-produced mammary gland carcinogenicity, whereas tryptophan was without effect.

Regulation of heme metabolism and cytochrome p-450 levels in primary culture of rat hepatocytes in a defined medium

Liver cells were prepared from adult Sprague-Dawley rats and used for the determination of delta-aminolevulinic acid synthetase (ALAS) activity and cytochrome P-450 concentrations at different time intervals in tissue culture in a serum-free synthetic medium. During the first 24 hr in culture, the level of cytochrome P-450 decreased to 30-40% of the level in isolated liver cells from untreated animals. The disappearance of cytochrome P-450 was especially fast in hepatocytes obtained from female phenobarbital-treated rats where only 40% of the original cytochrome P-450 was present after 2 hr in culture and 80% had disappeared in 2 days. The activity of ALAS increased 3- to 4-fold when measured 2 hr after plating, and it reached the maximum level in 19-24 hr when its activity was about eight times the original activity. In 2-4 days in culture, the activity of ALAS was four to five times above the original level. When the amount of delta-aminolevulinic acid (ALA) in the medium was increased from 1 to 100 microM, a decrease in ALAS was obtained, but no significant increase in cytochrome P-450 level was observed. Addition of heme to the medium gave a dose-dependent decrease in the activity of ALAS. Our data indicate that during the first 24 hr in culture the increase of ALAS activity was prevented by exogenous heme. This effect may be due to inhibition of the catalytic activity, suppression of the synthesis of the enzyme, or accelerated breakdown of the enzyme by heme.

2,4-diaminoanisole-induced thyroid pigmentation in rats inhibited by m-phenylenediamine

The effect of the carcinogenic hair dye component 2,4-diaminoanisole (2,4-DAA) on thyroid and pituitary morphology was studied. Heavy pigmentation of the hypertrophied thyroid epithelium was present when the animals were fed with 2,4-DAA for 6 weeks. Another hair dye component m-phenylenediamine (m-PDA), which lacks the methoxy group, and which is not carcinogenic, prevented the dark pigmentation of the thyroid epithelium caused by 2,4-DAA, but had only a slight effect on the hypertrophy of the gland. In the pituitary gland of 2,4-DAA-fed animals only a few aldehyde fuchsin positive thyrotrope cells were present. When 2,4-DAA and m-PDA were fed simultaneously, the number of hypertrophied chromophobic cells was greatly increased and the aldehyde fuchsin-positive cells were practically absent.

The effect of hydroxylamine on the morphology of the rat mammary gland and on the induction of mammary tumors by 7,12-dimethylbenz[a]anthracene

Abstract The effect of hydroxylamine (HA) on the morphology of the rat mammary gland and on the formation of mammory tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) was studied. Hydroxylamine caused excessive growth and secretory activity in the mammary gland. When it was given after DMBA administration it decreased the number and size of tumors per tumor bearing animal, but increased the median latency period. When HA was given before carcinogen administration it did not affect the incidence of tumors or the number or weight of tumors per tumor bearing animal, but it protected to some extent the normal lobular structure of the gland against the DMBA induced destruction which was evident a few weeks after carcinogen administration. A delayed effect of DMBA treatment was the formation of dark-staining irregular structures which originated either from the larger ducts or from the duct terminals. These formations were morphologically different from the hyperplastic alveolar nodules (HAN) which showed distended ductules of regular shapes and were common in animals treated with HA before DMBA administration.

Cellular Biology of the Rat Hepatic Stem Cell Compartment

The existence of hepatic stem cells has been, and no doubt will continue to be, a matter of considerable controversy. This controversy is partly fueled by the fact that cell turnover in the liver is very slow and the two major types of hepatic epithelial cells, hepatocytes and biliary epithelia, are capable of proliferation and can, at least in a healthy liver, meet replacement demands of cellular loss from these two differentiated populations. The best example of the capacity of adult hepatocytes and bile epithelial cells to proliferate is seen after partial hepatectomy in rats and mice, in which the compensatory hyperplasia of these cells in the remaining lobes restore the liver mass. The increased use and success of liver transplantation in clinical medicine have shown that these animal models correctly reflect the capacity of the human liver to regenerate (Van Thiel et al., 1989). What then is the evidence that there exists a stem cell compartment in the liver? The existence of hepatic stem cells was first postulated by Wilson and Leduc in 1958 based on experiments involving liver regeneration in the mouse after chronic injury induced with a methionine-rich basal diet mixed with an equal amount of bentonite (Wilson and Leduc, 1958). The authors concluded that “prolonged and severe injury to the liver may make direct restoration by division of pre-existing parenchymal cells impossible, and that, when this occurs, the new parenchyma is derived from the indifferent cholangiole cells.”

In vitro effect of L-tryptophan and its metabolites on dimethylaminoazobenzene reductase activity of rat liver

Abstract The exact role of azodye reductase, a liver microsmal enzyme, and its influence on the hepatocarcinogenicity of 4-dimethylaminoazobenzene (DAB) are uncertain. The effect of DAB is depressed by many nutritional factors, including trytophan. Therefore, the effects of o-aminophenol and of L -tryptophan and its metabolites L -kynurenine, anthranilic acid, kynurenic acid, quinaldic acid, 3-hydroxy- DL -kynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, quinolinic acid, N-methylnicotinamide, and N′-methylnicotin-amide on rat liver azoreductase activity were determined, using DAB as substrate. Only 3-hydroxyanthran- ilic acid, 3-hydroxykynurenine and o-aminophenol decreased enzyme activity. The inhibition was greater if the buffered solutions (pH 7.4) of these three compounds were kept overnight before use, but the effect was prevented if these compounds were prepared in solutions of L -ascorbic acid and/or L -cysteine HCl. This observation indicates that the autoxidation products were probably responsible for inhibition of the enzyme. Further study of the oxidation products including the phenylquinoneimine formed from the oxidation of 3-hydroxyanthranilic acid in air, cinnabarinic acid, xanthommatin, 2-amino-3H-isophenoxazin-3-one, 1,9-dimethyl-2-amino-3H-phenoxazin-3-one and actinomycin D showed that all these compounds inhibited enzyme activity. A non-competitive type of inhibition was observed in the presence of cinnabarinic acid and xanthommatin. Cinnabarinic acid, xanthommatin, 2-amino-3H-isophenoxazin-3-one, 1,9-dimethyl-2-amino-3H-phenoxazin-3-one, a nd actinomycin D all have the same phenoxazinone ring system, suggesting that the driving factor in the inhibition of the azoreductase is the presence of the phenoxazinone chromophore. The chemical resemblance of these phenoxazinones to the coenzyme riboflavine further supports this supposition.

Dimethylnitrosamine demethylase activity of rat liver microsomes after partial hepatectomy.

Abstract It is known that partial hepatectomy increases the hepatocarcinogenicity of dimethylnitrosamine (DMN). To investigate why this procedure increases the hepatocarcinogenicity of DMN, the activity of liver DMN demethylase was determined on Wistar male rats at different time intervals after partial hepatectomy. The ld 50 for DMN administered 1 day after partial hepatectomy was also compared to that for the control animals. The enzyme activity reached its lowest point (47 per cent) 1 day after operation, and at 3 days had recovered to about 90 per cent of the control values. The ld 50 for partially hepatectomized rats 44 hr after i.p. injection of DMN was 114 mg/kg body weight as compared to 82 mg/kg for control animals. The reduction rather than increase of enzyme activity after hepatectomy shows that increased DMN carcinogenicity after hepatectomy is not caused by a compensatory increase of demethylase activity associated with liver regeneration.