Toxicogenomic analysis of N-nitrosomorpholine induced changes in rat liver: comparison of genomic and proteomic responses and anchoring to histopathological parameters.

A common animal model of chemical hepatocarcinogenesis was used to examine the utility of transcriptomic and proteomic data to identify early biomarkers related to chemically induced carcinogenesis. N-nitrosomorpholine, a frequently used genotoxic model carcinogen, was applied via drinking water at 120 mg/L to male Wistar rats for 7 weeks followed by an exposure-free period of 43 weeks. Seven specimens of each treatment group (untreated control and 120 mg/L N-nitrosomorpholine in drinking water) were sacrificed at nine time points during and after N-nitrosomorpholine treatment. Individual samples from the liver were prepared for histological and toxicogenomic analyses. For histological detection of preneoplastic and neoplastic tissue areas, sections were stained using antibodies against the placental form of glutathione-S-transferase (GST-P). Gene and protein expression profiles of liver tissue homogenates were analyzed using RG-U34A Affymetrix rat gene chips and two-dimensional gel electrophoresis-based proteomics, respectively. In order to compare results obtained by histopathology, transcriptomics and proteomics, GST-P-stained liver sections were evaluated morphometrically, which revealed a parallel time course of the area fraction of preneoplastic lesions and gene plus protein expression patterns. On the transcriptional level, an increase of hepatic GST-P expression was detectable as early as 3 weeks after study onset. Comparing deregulated genes and proteins, eight species were identified which showed a corresponding expression profile on both expression levels. Functional analysis suggests that these genes and corresponding proteins may be useful as biomarkers of early hepatocarcinogenesis.

Sequential hepatic histologic and histochemical changes produced by diethylnitrosamine in the rhesus monkey.

Six young adult male rhesus monkeys were given diethylnitrosamine ip for 3-5 years. Liver biospies were done monthly. After 6 months, biopsy specimens showed individual hepatocytes and small foci of hepatocytes that were intensely positive for glycogen. During the second and later years, larger foci of such cells developed. In sections stained with hematoxylin and eosin, the glycogen-containing hepatocytes generally appeared unusually clear. Some hepatocytes, however, had eosinophilic or basophilic cytoplasm. Nuclear enlargement and atypic developed, particularly outside the foci. The hepatocytes within most foci were uniform in their histochemical features: glycogen was elevated, glucose-6-phosphatase was decreased, and ATPase activity was present not only along the bile canalicular surface but also along the enire cell membrane. After 3-5 years, neoplastic nodules and hepatocarcinomas developed in 5 of 6 animals. Two nodules and particularly the heptocarcinomas differed from the foci in one of more histochemical parameters. The findings suggested that the glycogen-containing, histochemically altered cells of the foci in one or more histochemical parameters. The findings suggested that the glycogen-containing, histochemically altered cells of the foci may be the first step in the development of neoplasia; further steps toward malignancy appeared to be frequently associated with additional alterations, such as loss of sinusoidal ATPase and re-formation of glucose-6-phosphatase.

Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodules.

Cytochemical and electron microscopic investigations of neoplastic nodules induced in the rat liver by nitrosomorpholine or thioacetamide show that most neoplastic nodules are comprised of a rather heterogeneous cell population. At least four different types of altered hepatocytes can be distinguished: (a) "clear" glycogen storage cells with a dislocation and relative reduction of the granular endoplasmic reticulum; (b) "acidophilic" glycogen storage cells with a hypertrophy of the agranular endoplasmic reticulum; (c) fat-storing cells; and (d) basophilic cells poor in glycogen and rich in ribosomes. In addition, there are diverse intermediate cell types. The cytochemically demonstrable activity of glucose-6-phosphatase is reduced in most neoplastic nodules, but it may also be normal or even increased. The clear and the acidophilic cells precede the development of the neoplastic nodules by weeks and months. They usually form foci which are taken to be preneoplastic lesions. During the formation of neoplastic nodules and hepatocellular carcinomas originating for such foci the glycogen of the clear and the acidophilic cells is progressively reduced, whereas the number of ribosomes (basophilia) increases. This process, which may be accompanied by a transitory accumulation of fat, leads to the evolution of basophilic carcinoma cells. We conclude from these observations that the majority of the neoplastic nodules consist of a mixture of precancerous, definitely cancerous, and diverse intermediate cells. Neoplastic nodules in which basophilic cells prevail may already be carcinomas. Although the neoplastic nodules seem to be a frequent precursor of hepatocellular carcinomas, the latter may also develop without going through the nodule stage.

Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.

Expression of two important glucose transporter proteins, GLUT 2 (which is the typical glucose transporter in hepatocytes of adult liver) and the erythroid/brain type glucose transporter GLUT 1 (representing the typical glucose transporter in fetal liver parenchyma), was studied immunocytochemically during hepatocarcinogenesis in rats at different time points between 7 and 65 wk after cessation of 7-wk administration of 12 mg/kg of body weight of N-nitrosomorpholine p.o. (stop model). Foci of altered hepatocytes excessively storing glycogen (GSF) and mixed cell foci (MCF) composed of both glycogenotic and glycogen-poor cells were present at all time points studied. Seven wk after withdrawal of the carcinogen, GSF were the predominant type of focus of altered hepatocytes. Morphometrical evaluation of the focal lesions revealed that the number and volume fraction of GSF increased steadily until Wk 65. MCF were rare at 7 wk, increased slightly in number and size until Wk 37, but showed a pronounced elevation in their number and volume fraction from Wk 37 to Wk 65. In both GSF and MCF, GLUT 2 was generally decreased or partially absent at all time points. Consequently, foci of decreased GLUT 2 expression showed a steady increase in number and volume fraction from Wk 7 to Wk 65. GLUT 1 was lacking in GSF but occurred in some MCF from Wk 50 onward. The liver type glucose transporter GLUT 2 was decreased in all adenomas and hepatocellular carcinomas (HCC). In three of seven adenomas and 10 of 12 carcinomas, expression of GLUT 1 was increased compared with normal liver parenchyma. In two cases of adenoid HCC, cells of ductular formations coexpressed GLUT 2 and GLUT 1. In contrast, normal bile ducts, bile duct proliferations, and cystic cholangiomas expressed only GLUT 1. Seven of 12 HCC contained many microvessels intensely stained for GLUT 1, a phenomenon never observed in normal liver. Whenever adenoid tumor formations occurred, GLUT 1-positive microvessels were located in the immediate vicinity of these formations. Only in one HCC were such microvessels found in the absence of adenoid formations. Our studies indicate that a reduction of GLUT 2 expression occurs already in early preneoplastic hepatic foci and is maintained throughout hepatocarcinogenesis, including benign and malignant neoplasms. Reexpression of GLUT 1, however, appears in a few MCF and in the majority of adenomas and carcinomas.

Histochemical characterization of focal hepatic lesions induced by single diethylnitrosamine treatment in infant mice.

Focal hepatocellular lesions, induced in our infant mouse system (15-day-old B6C3F1 mice) by a single carcinogenic dose of diethylnitrosamine (2.5 or 5.0 micrograms/g body weight), were characterized histochemically using toluidine blue, periodic acid-Schiff, glycogen phosphorylase, glycogen synthetase, glucose-6-phosphatase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, ATPase, gamma-glutamyl transpeptidase, and acid phosphatase. Animals were killed 5, 12, 18, and 24 weeks following diethylnitrosamine treatment. The first focal lesions were observed in mice killed at 12 weeks. All foci showed patchy cytoplasmic basophilia and a slight decrease in the glycogen content. The early foci (12 weeks) showed no change in the levels of glycogen phosphorylase and glycogen synthetase, a strong reduction of glucose-6-phosphatase, and a high increase in glucose-6-phosphate dehydrogenase. In addition, 56% of foci in males and 86% of foci in females showed a slight rise in glyceraldehyde-3-phosphate dehydrogenase, and 12% of foci in males and 17% of foci in females had a lower acid phosphatase. The level of cytoplasmic ATPase was slightly decreased in 22% of foci. By 24 weeks, a decrease in the activity of cytoplasmic ATPase was observed in 84 and 100% of foci in males and females, respectively. The increase in the membrane ATPase was observed in 65% of foci in males and 7% of foci in females. By that time, the decrease in acid phosphatase was observed in 78% of foci in males and 37% of foci in females. The gamma-glutamyl transpeptidase failed to show any increase in its activity, indicating that this enzyme was not a "marker" of the hepatocellular lesions developing under the experimental conditions. Strong decrease in glucose-6-phosphatase in association with a manifest increase in glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activities indicated a shift from gluconeogenesis to glycolysis. Since this metabolic shift occurred concurrently with an increase in the labeling indices and focal size, it appears that these changes act in concert, representing expression of the acquired functional and replicating potential of the focal cell population.

Histochemical analysis of the development of estradiol-induced kidney tumors in male Syrian hamsters.

Chronic administration of the estrogen 17 beta-estradiol induces kidney tumors in male Syrian hamsters within 6 months of initial exposure. Although these tumors have previously been studied histologically and histochemically and have been postulated to be derived from proximal tubular and/or interstitial cells, there exists no unambiguous evidence for an epithelial or mesenchymal origin. To elucidate the histogenesis of these neoplasms, kidney sections of hamsters treated with estradiol for 4, 5, and 6 months and age-matched untreated controls were investigated histologically and histochemically. Proliferating foci were observed in kidneys exposed to estradiol for 5 and 6 months. They consisted of clusters of spindle-shaped cells forming solid blocks, cords, or branches located between tubules. These foci were judged to be precursors of larger tumors identified in the latter treatment group. The histological and histochemical profile of foci and tumors matched closely. These lesions were marked by very high activities of alkaline phosphatase, adenyl cyclase, and glucose 6-phosphate dehydrogenase. In contrast, glycogen content and activities of glucose 6-phosphatase, succinate dehydrogenase, and gamma-glutamyl transpeptidase were low or absent. Immunofluorescence of the intermediate filaments revealed that foci and tumors solely expressed vimentin and desmin but not cytokeratin. The morphology, enzyme histochemical pattern, and immunofluorescence strongly support a mesenchymal origin of the estradiol-induced hamster kidney tumors studied. The neoplasms were probably derived from vascular smooth muscle cells of a cell subtype particularly sensitive to hormonal stimulation and transformation.

Histochemical profile of mouse hepatocellular adenomas and carcinomas induced by a single dose of diethylnitrosamine.

In continuation of earlier studies on murine neoplastic liver lesions, we characterized by histochemical methods the phenotype of hepatocellular adenomas and carcinomas induced by single injections of diethylnitrosamine (1.25, 2.5, or 5.0 micrograms/g of body weight) in 15-day-old C57BL/6 x male C3H F1 mice. The hepatocellular adenomas were composed predominantly of basophilic cells but stored excessive amounts of fat and glycogen in large portions of the tumors. Irrespective of the carcinogenic dose, the adenomas showed a consistent histochemical pattern. Glycogen synthase and phosphorylase were highly active in the hepatocytes that stored glycogen. In cells poor in, or free of, this polysaccharide, these enzymes were only moderately active or even inactive. In glycogen-storing parts of the adenomas, the activity of adenylate cyclase was reduced compared with normal liver parenchyma, but in fat-storing portions it was elevated. In a few adenomas, uniform increase in adenylate cyclase activity could be encountered. The levels of ATPase, acid phosphatase, and glucose-6-phosphatase were either increased or decreased. Glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase showed an increased activity in all adenomas compared with preneoplastic foci, which in turn exhibited a higher glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase activity than the surrounding parenchyma or the liver of untreated controls. The hepatocellular carcinomas showed remarkable histochemical changes compared with adenomas. The levels of fat and glycogen and the activities of glycogen synthase, phosphorylase, and in most cases also that of glucose-6-phosphate dehydrogenase, were reduced significantly. In contrast, adenylate cyclase, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase, and also alkaline phosphatase showed a striking elevation in developing carcinomas. Similar, although more pronounced, histochemical changes were seen in the advanced hepatocellular carcinomas. These observations indicated that progression from adenomas to hepatocellular carcinomas was associated with a change in the activity of several enzymes involved in cell membrane function, glycogen metabolism, the oxidative pentose phosphate pathway, and glycolysis.

Separation in distinct subpopulations by elutriation of liver cells following exposure of rats to N-nitrosomorpholine.

N-Nitrosomorpholine, administered with drinking water to SD rats at the daily dose of 2.4 mg/kg for 7 weeks, induces persisting changes in the hepatocytes as shown by electron microscopy and cytochemistry. In situ, the hepatocytes exhibit a heterogeneous reaction for glucose-6-phosphatase activity. Cells of large diameter, frequently deficient in this enzyme, contain a well-developed rough and/or smooth endoplasmic reticulum. Adult rat hepatocytes from control and N-nitrosomorpholine-treated rats were isolated by enzymatic perfusion. Isolated cell populations in both experimental models were composed of a few contaminating sinusoidal cells; small, intermediate, and large hepatocytes; and doublets or triplets of undissociated cells. Five distinct hepatic subpopulations were separated by elutraition or counterflow centrifugation and analyzed by morphological, morphometric, and cytophotometric methods. Fraction I is composed of small (16 to 18 micrometers) diploid hepatocytes; Fractions II and III consist of homogeneous populations of tetraploid cells (mean diameters, 20.5 and 22.4 micrometers); Fraction IV is enriched with large octoploid cells whose mean diameters reach 25.2 micrometers; and Fraction V contains large cells and cell aggregates. The counterflow centrifugation shows the higher proportion of hypertrophied and polyploid hepatocytes, obtained after carcinogen treatment, in the elutriated Fractions IV and V. The structural integrity of hepatocytes is not affected by the process of elutriation. Large hepatocytes, up to 30 micrometers in diameter, exhibit an abundant smooth endoplasmic reticulum, frequently disposed at the periphery of the cell where it forms a network of anastomosing tubules. Moreover, some of these cells present well-developed rough endoplasmic cisternae, closely associated in large fields. Under the scanning electron microscope, elutriated hepatocytes from control rats show numerous regularly distributed microvilli covering the entire cell surface, whereas hypertrophic hepatocytes from N-nitrosomorpholine-treated rats offer heterogeneous cell surfaces, characterized by the presence of patches of short, closely packed microvilli.

p53 Mutations in human hepatocellular carcinomas from Germany.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined 13 cases of human hepatocellular carcinomas from Germany for the presence of p53 aberrations in exons 4 to 8 of the gene by single-strand conformation polymorphism and restriction fragment-length polymorphism analyses and by sequencing of polymerase chain reaction products. Single base substitutions occurred in two human hepatocellular carcinomas: a C:G----T:A transition at a CpG site in codon 257, and a T:A----A:T transversion at codon 273. One of these point-mutated tumors and two additional tumors without point mutations demonstrated a loss of one p53 allele. None of the tumors was mutated in codons 12 or 61 of the c-Ha-ras gene.

Early detection of Knudson's two-hits in preneoplastic renal cells of the Eker rat model by the laser microdissection procedure.

Hereditary renal cell carcinomas invariably develop by the age of 1 year in Eker rats. At the histological level, renal cell carcinomas develop through multiple stages from early preneoplastic lesions (e.g., phenotypically altered tubules) to adenomas. We previously reported that ionizing radiation induces additional tumors (large adenomas and carcinomas) in a linear dose-response relationship and that loss of heterozygosity (LOH) at chromosome 10, where the predisposing tuberous sclerosis (Tsc2) gene is localized, was found in the renal cell carcinomas which developed from hybrid F1 rats carrying the Eker mutation, indicating that in heterozygotes two events (one inherited, one somatic) are necessary to produce at least large adenomas and carcinomas. This study was designed to examine LOH in the earliest preneoplastic lesions, using a laser microdissection procedure. We could accurately dissect single altered renal tubules out of freeze-dried sections and clearly detected LOH in 4 of 19 altered tubules (21%). This is the first demonstration of LOH in single renal tubules. Our present results support the theory of a second, somatic mutation (second hit) as rate-limiting step of renal carcinogenesis in the Eker rat model of dominantly inherited cancer and the tumor suppressor nature of the Tsc2 gene function.

Localization of estrogen receptors in interstitial cells of hamster kidney and in estradiol-induced renal tumors as evidence of the mesenchymal origin of this neoplasm.

The mechanism of estrogen-induced and -dependent kidney carcinogenesis in Syrian hamsters and the cell of origin of the tumor are not well understood; they have been investigated in this study by mapping the cellular locations of estrogen receptor (ER) in estrogen-dependent tumors, in kidney tissue of hamsters treated with estradiol for 0.5 and 5.5 months, and in kidneys of age-matched controls. To validate the methods used, receptors have also been localized in uteri of hamsters and rats and in female hamster kidneys. ERs have been identified in cryostat sections by immunocytochemical techniques using an affinity-purified ER antibody, ER-715. Nuclei of tumors were intensely stained for ERs. In estrogen-treated kidneys and in controls, ER protein was identified in interstitial cells and capillaries, in arteries, and in renal corpuscles, particularly in podocytes and in the parietal layers surrounding the renal corpuscles. There was no ER protein in tubular epithelia even when tubuli were surrounded by tumor cells. The ER distribution in female hamster kidneys closely matched that in male kidneys. However, the staining intensity was stronger in female than in male kidneys. In hamster uteri, there was an intense ER-positive reaction in the nuclei of stroma, in stromal vessels, and in the luminal epithelia as demonstrated previously by others in rat uteri. ER mRNA has also been demonstrated by Northern blot analysis in estrogen-treated kidneys which contained tumors but was undetectable in untreated kidneys. The localization of ERs in estrogen-dependent tumors and in interstitial cell types but not in tubular epithelia supports previous conclusions of an interstitial origin of estrogen-induced hamster kidney tumors.

Synergistic hepatocarcinogenic effect of hepadnaviral infection and dietary aflatoxin B1 in woodchucks.

Interactive hepadnaviral and chemical hepatocarcinogenesis was studied in woodchucks inoculated as newborns with woodchuck hepatitis virus (WHV), which is closely related to the human hepatitis B virus. When the woodchucks reached 12 months of age, aflatoxin B1 (AFB1) was administered in the diet at dose levels of 40 micrograms/kg body weight/day for 4 months and subsequently 20 micrograms/kg body weight/day (5 days/week) for lifetime. WHV DNA was demonstrated by Southern blot hybridization in the serum and by PCR in the serum and/or liver tissue. The histo- and cytomorphology of the liver were investigated by light and electron microscopy. WHV carriers with and without AFB1 treatment developed a high incidence of preneoplastic foci of altered hepatocytes, hepatocellular adenomas, and hepatocellular carcinomas that appeared 6-26 months after the beginning of the combination experiment. Administration of AFB1 to WHV carriers resulted in a significantly earlier appearance of hepatocellular neoplasms and a higher incidence of hepatocellular carcinomas compared to WHV carriers not treated with AFB1. Neither hepatocellular adenomas nor carcinomas (but preneoplastic foci of altered hepatocytes) were detected in woodchucks receiving AFB1 alone, and no preneoplastic or neoplastic lesions were found in untreated controls. These results provide conclusive evidence of a synergistic hepatocarcinogenic effect of hepadnaviral infection and dietary AFB1. Except for the frequent presence of ground glass cells containing surface antigen filaments in the infected woodchucks, the phenotype of preneoplastic foci of altered hepatocytes was similar in WHV carriers with and without exposure to AFB1 and in animals treated with AFB1 alone. Clear cell foci excessively storing glycogen and/or fat, amphophilic cell foci crowded with mitochondria and peroxisomes, and mixed cell foci composed of various cell types including basophilic cells rich in ribosomes predominated. The cellular phenotype in neoplastic lesions varied from clear, amphophilic, and mixed cell populations in highly differentiated adenomas and carcinomas to basophilic cell populations prevailing in poorly differentiated carcinomas. The striking similarities in altered cellular phenotypes of preneoplastic hepatic foci emerging after both hepadnaviral infection and exposure to AFB1 suggest closely related underlying molecular mechanisms that may be mainly responsible for the synergistic hepatocarcinogenic effect of these oncogenic agents.

Differences in expression and intracellular distribution of hexokinase isoenzymes in rat liver cells of different transformation stages.

The activity, intracellular distribution and mRNA expression of hexokinase isoenzymes were studied in normal rat liver, and in epithelial liver cells at different stages of neoplastic transformation, including non-tumorigenic and tumorigenic cell lines. In contrast to liver, all transformed cells exhibited only hexokinase I and II, which both showed significantly increased activity, hexokinase II being the more abundant form. In parallel, the mRNA expression of the two isoenzymes was elevated, indicating transcriptional control of gene expression. Hexokinase I and II were found in the cytosol and bound to mitochondrial membranes; the percentage of membrane-bound enzyme activity increased with the grade of transformation from 32% of total activity in normal liver up to 69% in dedifferentiated tumor cells. The ratio of hexokinase I/II was higher in the membrane fraction than in the cytosol. In all tissues studied hexokinase II could be resolved in two subtypes IIa and IIb by hydrophobic interaction chromatography. The relative proportion of cytosolic IIa and IIb varied significantly between normal liver (1:1) and transformed cells, and among cells of different transformation stages (4:1 to 1:10). IIa demonstrated the main activity in the more differentiated, IIb in the less differentiated cell lines. IIa-activity showed a good correlation with the intracellular glucose 6-phosphate concentration of the cells. The data indicate that neoplastic cell transformation is accompanied by progressive alterations in the proportion and subcellular distribution of hexokinase isoenzymes I and II.

Increase of lipid peroxidation in rat liver microsomes by dehydroepiandrosterone feeding.

Oral administration of the adrenal steroid dehydroepiandrosterone (DHEA), a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in microsomes isolated from rat liver and kidney cortex, but not from brain. The increase of liver microsomal lipid peroxidation was greater in male than in female rats. the effect of DHEA on lipid peroxidation became discernible after feeding steroid-containing diet (0.6%) to male and female rats for 2 and 3 days and reached maximal levels at 1 and 2 weeks, respectively. The increase of microsomal lipid peroxidation reached a plateau stimulation at 0.05% in the diet. The addition of DHEA in the concentration range 0.1-100 microM to microsomes isolated from control rats had no effect on lipid peroxidation. Furthermore, a significant increase of the endogenous concentration of thiobarbituric acid reactive substances was found in microsomes after DHEA-administration at 0.05% in the diet. These results provide in vivo evidence that DHEA can cause lipid peroxidation in rat liver. Administration of DHEA at 0.6% in the diet for 7 consecutive days also significantly enhanced NADH- and ascorbate-dependent lipid peroxidation in liver microsomes. The DHEA-stimulated rat liver microsomal lipid peroxidation was completely inhibited by EDTA but not by superoxide dismutase, catalase or mannitol applied as OH-radical scavenger. The findings indicate that membrane lipid peroxidation is an early effect of DHEA, and that this process may be involved in the steroid-induced carcinogenesis in rats.

Investigation of the carbohydrate metabolism of normal and neoplastic hepatocytes using 2,6-dichlorophenolindophenol as a probe for NAD(P)H production measured by voltammetry.

An electrochemical technique is described for measurement of intracellular NAD(P)H production. This technique involves an auxiliary redox system taken up by the cells which is then measured voltammetrically after reduction by NAD(P)H. The redox system used was 2, 6-dichlorophenolindophenol (DCPIP). It was shown to undergo a quasi-reversible two-electron transfer at the rotating gold disc electrode serving as an indicator electrode. The anodic wave of the reduced form of DCPIP was taken to indicate the amount of NAD(P)H produced by metabolic processes with glucose as substrate for a given number of cells. The following types of cell were investigated in suspension: Morris hepatoma 3924, a hepatocyte-derived cell line, and normal hepatocytes. Marked differences between normal and transformed cells were found under aerobic compared to anaerobic conditions. These were explained in terms of alterations in carbohydrate metabolism, e.g., Pasteur effect occurring on cell transformation.

Voltammetric measurements of the kinetics of enzymatic reduction of 2, 6-dichlorophenolindophenol in normal and neoplastic hepatocytes using glucose as substrate.

Amperometric methods were used to study reaction kinetics of 2, 6-dichlorophenolindophenol (DCPIP) with NADH in the homogeneous phase. The second-order rate constant of the reaction was calculated to be 2.4 M-1 . s-1 at 37 degrees C. The reoxidation of the reduced form of DCPIP in air-saturated phosphate-buffered saline was found to follow pseudo-first-order reaction kinetics with rate constant of 5.9 X 10(-4) s-1. These data were compared to the reduction of DCPIP in suspensions of normal and neoplastic hepatocytes, in the absence and presence of oxygen. The reduction of DCPIP by intracellular NAD(P)H was shown to follow mixed-type reaction kinetics different from those of the homogeneous phase. From this, the conclusion was drawn that complexes of enzymes transferring electrons to and from NAD(P)H are involved in intracellular reduction of DCPIP.

Evaluation of the kinetics of the intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes measured by amperometric methods.

Amperometric methods were used to study the kinetics of intracellular reduction of 2,6-dichlorophenolindophenol (DCIP) in normal and transformed hepatocytes with glucose and succinate as substrates. The curves showing the formation of DCIPred as a function of time were biphasic, the first part obeying the equation of a pseudo-first-order reaction, the final part corresponding to Michaelis-Menten kinetics. A statistical method was used to estimate pseudo-first-order rate constants k as well as Km and Vmax values. At saturating glucose concentrations k, Km and Vmax values were higher in normal compared to transformed cells. Decreasing glucose concentrations revealed lowered saturation concentrations in tumour cells compared to normal cells. With succinate as substrate for hepatocytes, k values were higher than with glucose, while Km and Vmax were about the same. Hepatoma cells did not metabolize succinate. K values could be attributed to intracellular dehydrogenase activities including cytosolic and mitochondrial processes. Differences in pseudo-first-order rate constants between normal and tumour cells may therefore represent characteristic alterations associated with transformation.

Investigation by amperometric methods of intracellular reduction of 2,6-dichlorophenolindophenol in normal and transformed hepatocytes in the presence of different inhibitors of cellular metabolism.

The reduction of 2,6-dichlorophenolindophenol (DCIP) was measured by amperometric methods in Morris hepatoma 3924A cells, normal isolated rat hepatocytes and in mitochondria isolated from normal rat liver. The influence of aerobic and anaerobic atmospheres and of various inhibitors of cellular metabolism, especially of the respiratory chain (KCN, NaN3, oligomycin), on DCIP-reduction were studied using glucose, succinate, beta-hydroxybutyrate, alpha-keto-glutarate and oxalacetate as substrates. Under the influence of KCN and oligomycin the velocity of DCIP-reduction was increased in both cell types. Azide showed a similar effect on tumour cells and to a lower extent on hepatocytes. Using isolated mitochondria total DCIPred was increased by KCN and azide using various mitochondrial metabolites as substrates and with ADP/Pi present. The effects of KCN, azide and oligomycin could be explained by taking DCIP as an artificial coupling site in mitochondria which is only used when oxygen is absent or when the respiratory chain is blocked by inhibitors of cytochrome oxidase. Evaluation of the reaction kinetics revealed differences between normal and transformed cells in terms of the pseudo-first-order rate constants and the activity of overall oxidoreductases. The results apparently reflect quantitative differences in enzymatic equipment and the metabolic pathways predominating in normal and neoplastic cells.

Mitochondrial anomalies in renal oncocytes induced in rat by N-nitrosomorpholine.

Renal oncocytes were induced in male Sprague-Dawley rats by oral application of N-nitrosomorpholine (NNM) in a concentration of 12 or 50 mg per 100 ml drinking water and analysed by transmission electron microscopy. The oncocytes exhibited mitochondrial anomalies which were very similar to those of human oncocytoma oncocytes. The most striking mitochondrial changes were an unusually large number of the organelles and an excess of the cristae mitochondriales. The shape of the mitochondria varied from round and oval to extremely elongated slender or cup-shaped organelles often piled up to form complex bodies. Rarely large ramifying mitochondria were found. In addition to transversely orientated cristae or tubules the inner mitochondrial membranes frequently formed dense stacks of unusually long cristae often arranged longitudinally. In some mitochondria straight and strictly parallelly arranged neighbouring cristae were found to be connected by a paracrystalline array of pillars bridging the mitochondrial matrix. In the majority of oncocytes the mitochondrial matrix contained prominent dense granules (diameter 30-50 nm), but sometimes these were rare or absent. Individual mitochondria of many oncocytes showed membrane-bound deposits of glycogen particles which usually formed rosettes (alpha-particles) with a diameter of about 80 nm. Partitioned mitochondria were not seen. Often autophagic vacuoles containing portions of degraded mitochondria and dense bodies, probably autophagosomes, were observed. The nature and significance of the oncocytes remains unclear, but we suggest from these and earlier experimental results that oncocytes represent a special type of a neoplastically transformed cell which usually has only a low potential for "autonomous" growth. Renal oncocytes induced in rats by chemical carcinogens appear to be an excellent experimental model for the further investigation of this cell type.

Spongiosis hepatis in rats treated with low doses of hepatotropic nitrosamines.

Spongiosis hepatis, a characteristic pathomorphologic entity originating from the perisinusoidal liver cells, was observed in rats after administration of relatively low doses of the hepatocarcinogens dimethylnitrosamine (DMNA) and nitrosopyrrolidine (NO-Pyr), but not after application of the urinary bladder carcinogen butyl-butanolnitrosamine (BBNA). The results support the conception that spongiosis is a persisting liver lesion due to specific changes of the perisinusoidal liver cells by hepatotropic carcinogens.