Rapid development of hyperplastic nodules and cirrhosis in the liver of rats treated concurrently with thioacetamide and the pyrrolizidine alkaloid lasiocarpine.

Pyrrolizidine alkaloids have long been considered to be hepatotoxic in man as well as in grazing animals. To investigate the effect of liver cell division induced by thioacetamide on the hepatic changes induced by these alkaloids, rats were treated concurrently with thioacetamide and the pyrrolizidine alkaloid lasiocarpine. Thioacetamide was given intraperitoneally in a dose of 50 mg/kg b. wt twice weekly and lasiocarpine was administered in the diet at a concentration of 50 ppm. At 15 weeks, the combination of thioacetamide and lasiocarpine produced numerous grossly visible grey nodules in livers of 26 of 30 rats. Microscopically, these livers revealed a severe degree of postnecrotic cirrhosis and numerous hyperplastic nodules. The cells in most nodules were arranged in solid sheets or in a trabecular pattern and showed atypia, mitosis and hyperchromasia. In contrast, there was no evidence of cirrhosis or nodule formation in livers of animals treated with either lasiocarpine or thioacetamide alone. The rapid development of liver lesions in rats treated simultaneously with low doses of lasiocarpine and thioacetamide suggests that cell proliferation accentuates the development of neoplasia.     

Hormonal regulation of sex differentiated parameters in liver nodules from rats treated in the resistant hepatocyte model

Sex differentiation of liver functions has been shown to beattenuated in preneoplastic rat liver nodules. The present studywas performed to investigate whether nodules from male ratsare to some extent withdrawn from the normal growth hormone(GH) regulation of these functions. Male and female Wistar ratswere treated according to a modified resistant hepatocyte model(RH-model), with diethylnitrosamine initiation and promotionwith intragastric administration of 2-acetylaminofluorene (2-AAF)combined with partial hepatectomy (PH). Eleven months post-initiationmale rats were treated with either human (hGH) or bovine growthhormone (bGH) or ovine prolactin (oPRL) by continuous infusionfor 1 week. The mRNA expression of a number of genes known tobe sex differentiated in liver from adult control rats was comparedin nodular and surrounding tissue from nodule-bearing male,female and hormone-treated male rats. The basal mRNA expressionof the female-predominant cytochrome P4502C12 (CYP2C12) wasincreased and the male-predominant CYP2C11 was decreased inliver nodules from male rats compared with the surrounding liver.Expression of the prolactin receptor (PRL-r; female > male)and the steroid 5-reductase (female > male) genes was decreasedin male nodules, whereas no difference was observed with respectto GHreceptor (GH-r; female > male) expression in nodulesversus surrounding tissue. Early nodules obtained from malestreated according to the original RH-model (dietary 2-AAF, 0.02%)and isolated 2 weeks after completion of the 2-AAF/PH treatmentshowed significantly lower GH-r mRNA levels than the total livertissue. In hepatocellular carcinomas from hormonally unmanipulatedmales 11 months post-initiation the decrease in PRL-r expressionwas even more pronounced than in the nodules and a significantdecrease in GH-r expression was seen. In female nodules theonly significant difference with respect to the sex differentiatedparameters was a lower 5-reductase expression than in the surroundingtissue. Continuous infusion of both hGH and bGH feminized theexpression of all the sex differentiated genes in male tissuesand eliminated the previously detected differences between nodulesand surrounding tissue. oPRL also eliminated the differencesbetween nodules and surrounding tissue in males and partly feminizedthe expression of both the 5reductase and the PRL-r genes. Althoughthe expression of several sex differentiated parameters in livernodules is altered compared with surrounding tissue, the preneoplasticlesions respond adequately to the feminizing effect of continuousGH infusion, thereby indicating that nodular tissue is not withdrawnfrom the normal endocrine control of rat liver.     

Rat hepatocyte nodules are resistant to the necrogenic effect of D-galactosamine.

D-Galactosamine is a known hepatotoxin which induces liver cell necrosis via depletion of UTP and other uridine nucleotides. Our previous work indicated that nodular hepatocytes have higher levels of total uridine nucleotides compared to normal liver, and in the present study we investigate the effect of galactosamine treatment on hepatocyte nodules and surrounding liver. Hepatic nodules were generated in male Wistar rats according to the Solt and Farber protocol. Six months after initiation animals received a single injection of D-galactosamine (500 mg/kg i.p.) and were then killed 1, 2, 4 or 7 days later. Histological analysis of liver revealed the presence of extensive liver cell necrosis in normal tissue 1 and 2 days after galactosamine treatment. However, very little or no necrosis was detectable inside hepatic nodules at any time point, indicating that these focal areas are resistant to the cytotoxic effect of galactosamine. This type of resistance could be the expression of a new component in the resistant phenotype of hepatic nodules.     

Genotoxicity of a variety of pyrrolizidine alkaloids in the hepatocyte primary culture-DNA repair test using rat, mouse, and hamster hepatocytes

Seventeen pyrrolizidine alkaloids were studied with the hepatocyte primary culture-DNA repair test using rat hepatocytes. DNA repair synthesis was elicited by 15 alkaloids, including 11 of unknown carcinogenicity, i.e., senecionine, seneciphylline, jacobine, epoxyseneciphylline, senecicannabine, acetylfukinotoxin, syneilesine, dihydroclivorine, ligularidine, neoligularidine, and ligularizine. The positive results with these alkaloids of unknown carcinogenicity suggest that they are possibly genotoxic carcinogens. The two pyrrolizidine alkaloids that did not elicit DNA repair were retronecine which lacks a necic acid component and ligularinine which lacks the unsaturated double bond at the 1,2-position of the pyrrolizidine ring. Five pyrrolizidine alkaloids, retronecine, monocrotaline, seneciphylline, senkirkine, and clivorine, were also tested in the DNA repair test with hamster or mouse hepatocytes. These alkaloids, except retronecine, showed a positive response in the test with hamster hepatocytes, but in the test with mouse hepatocytes clivorine in addition to retronecine was also negative. The results indicate a species difference in liver bioactivation of pyrrolizidine alkaloids, implying that there could be species differences in their carcinogenic activities.     

Role of cytochrome P450IIIA4 in the metabolism of the pyrrolizidine alkaloid senecionine in human liver

Studies were carried out to investigate the metabolism of senecionine by human liver microsomes and the role of human cytochrome P450IIIA4 in this process. Human liver microsomes metabolized senecionine to two major products, (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) and senecionine N-oxide. The rates of product formation (DHP and senecionine N-oxide) varied widely with the microsomal samples tested. There was a 30-fold difference in DHP formation and a 25-fold difference in N-oxidation between the poorest metabolizer and the highest metabolizer of senecionine. The conversion of senecionine to DHP and senecionine N-oxide in human liver microsomes was markedly inhibited by the mechanism-based inactivators of P450IIIA4, gestodene and triacetyloleandomycin. Anti-P450IIIA4 IgG, at a concentration of 1 mg/nmol of P450, was found to inhibit completely the formation of DHP and senecionine N-oxide in human liver microsomes (HL101) having low activity toward senecionine. At 5 mg IgG/nmol P450, anti-P450IIIA4 inhibited 90 and 84% respectively of the formation of DHP and senecionine N-oxide in liver microsomes (HL110) with the highest activity toward senecionine. The formation of DHP or senecionine N-oxide was highly correlated with the amount of P450IIIA4 measured in the microsomes using polyclonal anti-P450IIIA4 IgG. The rate of DHP production also had a strong correlation with the rate of senecionine N-oxide formation (r = 0.999) and with the rate of nifedipine oxidation (r = 0.998). Our present studies provide evidence that P450IIIA4 is the major enzyme catalyzing the bioactivation (DHP formation) and detoxication (senecionine N-oxide formation) of senecionine in human liver.     

Aldehyde dehydrogenase activities in hepatocyte nodules and hepatocellular carcinomas from Wistar rats

The activities of aldehyde dehydrogenases using benzaldehydeand propionaldehyde as substrates and NADP and NAD as coenzymeswere determined in normal liver, hepatocyte nodules and hepatocellularcarcinomas from male Wistar rats. Hepatocyte nodules were producedby intermittent exposure of rats to 0.05% 2-acetylaminofluoreneor by initiation with diethylnitrosamine followed by selectionusing 2 weeks of dietary exposure to 0.02% 2-acetylaminofluoreneand partial hepatectomy. The activities of propionaldehyde:NAD and benzaldehyde: NADP aldehyde dehydrogenases were increasedin hepatocyte nodules of all types as well as in most hepatocellularcarcinomas. The most prominent elevation of enzyme activitywas found in the cytosol of persistent hepatocyte nodules (35–60times) and some hepatocellular carcinomas (92 times) using benzaldehydeand NADP. The benzaldehyde: NADP aldehyde dehydrogenase activityvaried considerably between different nodules suggesting theexistence of a subpopulation of hepatocyte nodules with veryhigh enzymatic activities. The activity of propionaldehyde:NAD aldehyde dehydrogenase activity as well as of -glutamyltransferasedid not show substantial internodular variations. The activityof benzaldehyde: NADP aldehyde dehydrogenase in individual carcinomasinvestigated in these experiments varied extensively. The datadid not support the idea that all hepatomas had been developedfrom pre-neoplastic nodules with very high activity of thisenzyme.     

Formation of DHP-derived DNA adducts from metabolic activation of the prototype heliotridine-type pyrrolizidine alkaloid, lasiocarpine

Pyrrolizidine alkaloids (PAs) are probably the most common poisonous plants affecting livestock, wildlife, and humans. The PAs that have been found to be tumorigenic in experimental animals belong to the retronecine-, heliotridine-, and otonecine-type PAs. Our recent mechanistic studies indicated that riddelliine, a tumorigenic retronecine-type PA, induced tumors via a genotoxic mechanism mediated by the formation of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts. The same adducts were formed from clivorine, a tumorigenic otonecine-type PA from metabolism of clivorine by rat liver microsomes in the presence of calf thymus DNA. In this study, we report that metabolism of lasiocarpine, the prototype heliotridine PA, by F344 rat liver microsomes resulted in the formation of DHP. When incubated in the presence of calf thymus DNA, the same DHP-derived DNA adducts were formed. These results suggest that these DHP-derived DNA adducts are potential biomarkers of exposure and tumorigenicity for all types of PAs.     

Pancreatic islet-cell and other tumors in rats given heliotrine, a monoester pyrrolizidine alkaloid, and nicotinamide.

Three rats of six males, surviving 22 to 27.5 months after one or two intragastric doses of the monoester pyrrolizidine alkaloid, heliotrine (230 mg/kg body weight), and pretreatment with nicotinamide (350 mg/kg body weight) by pretreatment with nicotinamide (350 mg/kg body weight) by i.p. injections, developed pancreatic islet-cell tumors, accompanied in one of the rats by transitory cell papillomas of the urinary bladder and interstitial testicular tumors and in another by a hepatoma. The lesions in the livers showed progression from megalocytosis, to microscopic hepatocellular hyperplasia, to increasingly larger nodules and hepatoma. One rat, given heliotrine, but no nicotinamide, also developed adenoma of the pancreatic islet cells. Adenomas of the pituitary were present among the experimental and also among the control rats killed between 19 and 27.5 months after the beginning of the experiment, and they are not likely to have been caused by the alkaloid. Heliotrine, in which the crucial double bond in the pyrrolizidine moiety is sterically hindered, appears to be less readily sequestered by the liver and also to affect other organs. Alkylation of nicotinamide at the N-1 position prevents its being reused for coenzyme biosynthesis. Hence, pretreatment of rats with large doses of nicotinamide prevents the depletion of nicotinamide adenine dinucleotide coenzymes and liver necrosis in rats given heliotrine (230 mg/kg body weight).     

The resistant hepatocyte model of carcinogenesis in the rat: the apparent independent development of oval cell proliferation and early nodules

The early cellular changes in the Solt-Farber resistant hepatocytemodel of carcinogenesis have been studied to clarify the relationshipof oval cell proliferation to the development of early hepatocytenodules. Cellular proliferation, intermediate filament profilesand the expression of specific cytochrome P450 enzymes wereexamined. At 24 h after partial hepatectomy (PH) many of thebile ductular cells were in S phase, but over the next few daysDNA synthesis progressively decreased in the portal bile ductsand was more common in arborizing ductules (oval cells) radiatingfrom the portal areas. These cells strongly expressed cytokeratins8 and 19 and vimentin, and from 1 week after PH they frequentlyunderwent differentiation either into hepatocytes, expressingcytochrome P450 enzymes, or into intestinal-type cells. Fivedays after PH, numerous basophilic foci were discernible, andthese expanded rapidly. The ductular cells swirled around thefoci, but their antigenic profile clearly indicated that thesecells were not involved in the development of these early nodules.In normal hepatocytes, cytokeratin 8 immunoreactivity was distinctlymembranous in location, and could only be readily detected inperiportal hepatocytes. In the basophilic hepatocyte foci, overexpressionof cytokeratin 8 was consistently associated with cells organizinginto acini, with expression reminiscent of authentic bile ducts,possibly indicating a structure-function relationship. In conclusion,early foci and nodules in this model are derived from resistanthepatocytes and not ductular oval cells, the latter being afacultative multipotential stem cell compartment.     

Isolation and characterization of endoplasmic reticulum and Golgi apparatus from hepatocyte nodules in male wistar rats

A method is described for the isolation of endoplasmic reticulum and Golgi apparatus from hyperplastic liver nodules produced by discontinuous feeding of 2-acetylaminofluorene to male Wistar rats. The procedure involves three centrifugation steps and permits the separation of these cell components and their subfractions from the same sample of liver tissue as little as 1 g, wet weight. The fractions have been characterized by chemical, enzymatic, and morphological techniques and were found to be as pure as preparations from normal tissue. Furthermore, some of the characteristic histochemical features of hyperplastic liver nodules have been quantitated by biochemical methods in the fractions. Glucose-6-phosphatase activity in the endoplasmic reticulum subfractions of nodules is approximately 15% of the corresponding value in normal livers, whereas the activity of reduced nicotinamide adenine dinucleotide phosphate: cytochrome c reductase is reduced to 85% of the normal activity. The amount of cytochrome P-450 in nodular membranes as measured by differential spectroscopy is 25% of the control, indicating a decreased Phase I activity in drug metabolism. A 5-fold increase in cytosolic glutathione S-transferase activity without change in the corresponding microsomal activity was detected in hepatocyte nodules in rat liver. The activity of gamma-glutamyltransferase is increased more than 20-fold in all membrane fractions prepared from nodular tissue. The cytosolic activity, which is very low in the normal liver, is similarly increased more than 20-fold. The membrane-associated gamma-glutamyltransferase seems to be an integral membrane protein which cannot be washed away from the membranes. Chemically, membranes from nodules have phospholipid and cholesterol:protein ratios as found in membranes from normal liver tissue. However, the composition of individual phospholipids is changed with a 2-fold increase in nodular phosphatidylinositol and a slight decrease in phosphatidylcholine content in nodular membranes. The amount of endoplasmic reticulum membranes is of the same magnitude as in normal liver, although the smooth-surfaced component constitutes almost 60% of the isolated endoplasmic reticulum marker enzymes in nodules, compared with only 32% in preparations from normal tissue. The albumin contents of nodular and normal microsomal and Golgi membrane preparations are similar, indicating a normal synthesis of albumin by nodular tissue.     

The enhancing effect of fasting/refeeding on the growth of nodules selectable by the resistant hepatocyte model in rat liver

Caloric restriction causes a generalized decrease in growthrate and has been shown to delay the development of both spontaneousand induced neoplasia. In contrast to chronic food restriction,the extreme condition of fasting/refeeding is associated withan overall increase in cell turnover in several organs, includingliver, compared with regular feeding. The present study wastherefore designed to investigate the effect of complete foodwithdrawal followed by refeeding on the growth of hepatocytenodules in initiated rat liver. Male Fischer 344 rats were givena single dose of diethylnitrosamine (DEN, 200 mg/kg i.p.) andthen, starting 1 wk later, they were exposed to one or threecycles of fasting (3 days) followed by refeeding (11 days).The control group was fed continuously. Seven weeks after DENadministration all rats were subjected to the resistant hepatocytemodel (2-acetylaminofluorene coupled with CCl₄) and 2 weekslater 2/3 partial hepatectomy (PH) was performed. All animalswere killed 2 weeks after surgery. At PH rats given one cycleof fasting/refeeding had significantly larger glutathione S-transferase7–7-positive hepatic lesions compared with controls (meanarea 0.73 ± 0.04 versus 050 ± 0.05 mm², P <0.025; mean percent area 25.6 ± 3.2 versus 12.4 ±0.9, P < 0.005), while no significant change was observedin their number. The observed differences were more pronouncedwith three cycles of fasting/ refeeding. A similar pattern ofresults was obtained at the time of killing. It is concludedthat fasting/refeeding can exert a positive effect on the growthof rat hepatocyte foci and nodules, in contrast to the generalinhibitory effect on carcinogenesis caused by food restriction.     

Changes in progesterone binding and metabolism in liver microsomes from persistent hepatocyte nodules and hepatomas in male rats

Hepatocyte nodules that persist throughout chemical carcinogenesis are linked to carcinomas both as one site at which hepatomas are seen to arise and as a tissue which shows more than a dozen significant protein changes also found in liver cancers. In view of the differential stimulus to growth of these persistent nodules by progesterone, progesterone metabolism and binding to the microsomes of nodules and hepatomas were studied. Progesterone metabolizing enzyme activities in nodule microsomes showed striking shifts with a 42% decrease in 16 alpha-hydroxylase activity and a 2- to 3-fold increase in 6 beta-hydroxylase activity compared to control levels. Hepatomas had a dramatic 20-fold increase relative to nodules or controls in the reductive pathway for progesterone metabolism as measured by delta 4-5 alpha-hydrogenase activity. The rate and saturation of the specific binding of progesterone to microsomes of nodules and liver cancers were significantly decreased when compared either to the tissue surrounding the nodules or to their respective control microsomes. This change in progesterone binding of nodular microsomes may relate to an altered balance of progesterone content and its metabolites in the nodular cells or to alterations in the microsomal membrane binding site. The functional significance of reduced binding of progesterone for liver carcinogenesis is thus open to further inquiry.     

7-Glutathione-pyrrole and 7-cysteine-pyrrole are potential carcinogenic metabolites of pyrrolizidine alkaloids

Many pyrrolizidine alkaloids (PAs) are hepatotoxic, genotoxic, and carcinogenic phytochemicals. Metabolism of PAs in vivo generates four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts that have been proposed to be responsible for PA-induced liver tumor formation in rats. In this present study, we determined that the same set of DHP-DNA adducts was formed upon the incubation of 7-glutathione-DHP and 7-cysteine-DHP with cultured human hepatocarcinoma HepG2 cells. These results suggest that 7-glutathione-DHP and 7-cysteine-DHP are reactive metabolites of PAs that can bind to cellular DNA to form DHP-DNA adducts in HepG2 cells, and can potentially initiate liver tumor formation.     

Failure of glutathione to prevent liver cancer development in rats initiated with diethylnitrosamine in the resistant hepatocyte model

This study was undertaken to observe whether the administrationof reduced glutathione intragastrically to male Fischer 344rats during the precancerous steps of liver carcinogenesis hasany protective effect on the development of hepatocellular carcinoma.Hepatocyte nodules were induced in the liver with a single initiatingdose of diethylnitrosamine followed by selection of resistanthepatocytes to generate nodules by a two week exposure to dietary2-acetylamino-fluorene coupled with partial hepatectomy. Animalshad hepatocyte (‘hyperplastic’) nodules when examinedby iaparotomy at three months. At that time, the animals weredivided into two groups. One received daily intragastric giuta-thionefor 8 months while the other received no further treatment.An additional control group received only the selecting (promoting)regimen with no initiator or glutathione. At 12 months, theanimals receiving the initiator and promoter regimen had a 65%incidence of hepatocellular carcinoma and those receiving glutathionein addition bad a 71% incidence. Under these experimental conditions,the long term administration of glutathione appears to haveno observable influence on liver cancer development in thismodel.