[Promoting effect of liver regeneration in hepatocarcinogenesis initiated by diethylnitrosamine in the rat]. / Effet promoteur de la régénération hépatique sur l'hépatocancérogenèse initiée par la diéthylnitrosamine chez le rat.

Preneopliastic cells initiated by a subcarcinogenetic diethylnitrosamine administration must be submitted to subsequent promoting treatments to reach malignancy. In order to elucidate this promotion mechanism we compare the cell kinetics regeneration induced either by a two-thirds hepatectomy or by two successive one-third hepatectomies. The two-thirds hepatectomy triggers acute well marked circadian mitotic waves of high amplitude. But, during this regeneration the preneoplastic cells remain without any proliferative advantage and without promoting effect. The first one-third hepetectomy induces two mitotic circadian waves of low amplitude; the second one-third hepatectomy recrutes 92% of cells already dividing after the first operation. These two successive regenerations liberate an important selective growth of preneoplastic cells and the tumor incidence reaches 96%. These results suggest that preneoplastic cells are selectively promoted by successive mitotic cycles induced by low mitogenic stimulations instead of by an acute stimulation.

[Influence of the stress induced by partial hepatectomy in the circadian liver regeneration]. / Influence du stress induit par l'hépatectomie partielle sur la synchronisation circadienne de la régénération hépatique.

Regeneration induced by partial hepetectomy is synchronized by the circadian variation of corticosterone that inhibits periodically the DNA synthesis. But, the amplitude and the postoperative delay of the first regenerating wave vary according to the nycthemeral time of the surgery that induces a powerful stress corticosterone release. Partial hepatectomy performed in the morning inhibits the subsequent corticosterone wave and the first DNA synthesis wave occurs early with a low amplitude. The same operation performed in the evening does not change the subsequent corticosterone wave and the first DNA synthesis wave is delayed but amplified. These results disprove the methodology used by many investigators: they sacrifice all the animals at the same nycthemeral time and perform operations at various delays before sacrifices in order to avoid the circadian synchronization.

[Synchronizing effect of corticosterone's circadian rhythm on the DNA synthesis rate in the liver of the young rat]. / Effet synchroniseur du rythme circadien de la corticostérone sur la vitesse de synthèse du DNA dans le foie du jeune rat.

Post-natal liver proliferation is synchronized by corticosterone circadian variation from the 21st day of life in rats. After tritiated thymidine injection, we compare the labelling index, the specific radioactivity of DNA, the DNA synthesis rate, the plasma corticosterone in un-rhythmed rats at day 15 and in rhythmed rats at day 25. No variation of these parameters is found during the 15th nycthemeron. During the 25th nycthemeron, the corticosterone circadian wave peaking at 18 h is followed by the circadian variations of the 3 proliferation parameters peaking at 24 h.

[Deterministic differential equations in cytokinetics]. / Equations différentielles déterministes en cytocinétique.

Carcinogenesis is generally viewed as the result of a disrupted equilibrium between the processes of proliferation, differentiation, migration and loss. In cell kinetics, perturbation of fundamental kinetic parameters such as the mitotic index or the growth fraction may reflect a carcinogenic process. In this paper, we present a system of deterministic differential equations describing the dynamical evolution of a cell population. We show that this model can account for asynchronicity of cells and simulate the fraction of labelled mitoses experiment as well.

The tumor promoting effect of constant light exposure on diethylnitrosamine-induced hepatocarcinogenesis in rats.

The hypothesis that light-induced circadian clock suppression exerts a promoting effect on liver carcinogenesis was investigated in rats. Sixty-five male Wistar rats were given diethylnitrosamine (DEN, 10 mg/kg/day p.o.) for 6 weeks and were randomized into 3 groups. Rats from group 1 (N=20) received DEN only. Rats from group 2 (N=22) also received phenobarbital (pheno, 30 mg/rat/day p.o.) for 4 weeks as a promoting agent and rats from group 3 (N=23) were exposed to continuous light. Three months after starting DEN treatment, urinary 6-sulfatoxymelatonin (alphaMT6s) excretion, a marker of circadian clock function, had lost its circadian rhythmicity in the LL group, with a 4-fold lower 24 h mean than that found in the LDpheno and LD groups (8.0 +/- 3.2 ng/ml, 33.6 +/- 3.1 ng/ml and 34.3 +/- 2.4 ng/ml respectively; p from ANOVA <0.001). Laparotomy was then performed. The proportion of rats with macroscopic nodules on liver surface was 72% (LD group), 89% (LDpheno group) and 95% (LL group) (p from chi2 = 0.1). Nodules were more numerous and larger both in the LL group and in the LDpheno one as compared to the LD group (p from chi2 <0.05). All the rats died with hepatocellular carcinomas, with a median survival of 5 months, similar in all 3 groups. Light-induced circadian clock suppression exerted a promoting effect similar to that caused by phenobarbital in this model, yet through different effects on circadian system function.

The role of corticoids, adrenalectomy, phenobarbital and pentobarbital in the promotion of DEN-hepatocarcinogenesis.

This paper shows there is a good correlation between the disturbance of the proliferation control and the plasma Corticosterone circadian pattern on the one hand and the promotion of diethylnitrosamine (DEN) initiated precancerous lesions on the other. In normal rats, phenobarbital (PB) or adrenalectomy, which both decrease plasma corticosterone levels, induce chronic liver growth, enhance the mitotic response to partial Hepatectomy and eliminate the mitotic circadian rhythms. In rats treated by DEN, the same treatments increase the proliferation advantage of PAS positive precancerous cells and promote tumorogenesis. Daily corticosterone injections inhibits liver growth even after hepatectomy both in normal and DEN-treated rats. Under carcinogenic conditions (DEN for 6 weeks), corticoid injections inhibit the selective growth of precancerous cells and the death by cancer occurs later. Pentobarbital (PE), administrated at the same subsedative doses as PB, does not change plasma corticosterone levels nor the mitotic control and does not enhance the carcinogenesis initiated by DEN. The "promoting effect" is closely linked to the disturbance of the biological corticosterone activity that normally synchronizes the liver cell proliferation by temporary inhibition of DNA synthesis for some hours every day (14-16). The role played by corticosterone in the selective growth of precancerous foci (glycogenesis type I) is discussed in the light of the hypothesis (1-2) according to which modification of the carbohydrate metabolism is closely related to the process of hepatocarcinogenesis.

Exogenous spermine induces maturation of the liver in suckling rats.

In the present study, we investigated the effects of spermine on postnatal liver maturation in suckling rats. The animals were given spermine either per os (8 micromol) or by intraperitoneal injection (1 micromol), once daily for three or five days. The percentage of liver cells in different cell cycle phases and of diploid cells in the parenchyma was estimated. The protein content, ornithine aminotransferase (OAT) activity, and content of DNA polyamines and receptors for polymeric immunoglobulins (RPI) were also measured in liver extracts. The ingestion of spermine had the following effects: the percentage of the cells in S and G2M phases of the cell cycle diminished the percentage of diploid cells increased the content of polymeric immunoglobulin receptors increased; the OAT activity increased; the contents of putrescine and spermidine decreased and almost reached adult values; and the spermidine/spermine ratio became similar to that observed in the liver of adult rats. These phenomena were detected 40 hours after the beginning of oral spermine treatment. The intraperitoneal injection of spermine had no effect on the OAT activity, but it decreased the spermidine content and enhanced the spermine content. Our data demonstrated for the first time that dietary polyamines play a role in the initiation of liver postnatal maturation in suckling rats.

[Analysis by computer simulation of cell kinetics of the liver]. / Analyse par simulation sur ordinateur de la cinétique cellulaire du foie.

A computer simulation model of cell kinetics is established to represent the variations of rat hepatocyte populations in several circumstances: normal growth, circadian rythms, response to partial hepatectomy under different conditions. This model differs from usual mathematical models-sets of equations--by the use of digital simulation techniques. Each cell in the population is represented by a set of variables in the computer memory. When experiments are simulated, the values of these variables are modified step by step according to the hypotheses we want to test. Counts and statistics derived at each step from the simulation are then compared to experimental values, in order to assess the validity of our hypotheses. This procedure enables us to establish a minimal set of conditions and outside causal effects necessary to mimic the behaviour of the liver under experimental situations similar to those we simulate. Such modelisation suggests further work on the mechanisms governing cell kinetics in the normal liver and during cancerisation.

[Use of biodegradable microspheres in experimental anticancer therapy. Chronopharmacological application]. / Utilisation des microsphères biodégradables en thérapie anti-cancéreuse expérimentale. Application chronopharmacologique.

We describe the initial experimentations which show that is possible to chronomodulate the cisplatin liberation out of some microspheres. The goal is to generate, inside one tumeur embolished by those cisplatin loaded microspheres, some concentration peaks at the best tolerance time. The cancer is than more hit, by the high local anticancer drug concentration, and doses chronomodulations preserve the patient by following his tolerance. The experimentation on cancerous mice show that this technique could lead to great survival increases. Such a protocol might usefuly improve the anticancer therapy.

[Chronotolerance of corticosterone on DNA synthesis in young growing rats]. / Chronotolérance de la corticostérone sur la synthèse du DNA chez le jeune rat en croissance.

Plasma corticosterone and DNA synthesis were measured during 2 circadian periodes following the 25th day of rats. Corticosterone (5 mg/kg) injected at 05 h (1 h before the normal corticosterone bathyphase) inhibits and dissynchronizes the nycthemeral evolution of the two parameters during the two subsequent periods. The same corticosterone administration injected at 17 h (1 h before the normal corticosterone acrophase) inhibits the first DNA synthesis wave but both parameters are nycthemerally restored from the second period. In this last case, the area under the second DNA curve compensates the inhibition of the first wave. The results are discussed in the view of chronocorticotherapy recommended in patients.

Importance of cell kinetics rhythmicity for the control of cell proliferation and carcinogenesis in rat liver (review).

The circadian control of cell Proliferation and Differentiation has been studied principally in rat liver. The comparison between the differentiation by hepatic enzymes and the division by the cell cycle under various experimental conditions (postnatal maturation, regeneration after partial hepatectomy, adrenalectomy, corticosterone treatments etc.) leads to the following conclusions: Under physiological conditions, proliferation and differentiation activities present a mutually exclusive relationship with a specific circadian rhythm. For both functions, the circadian variation of corticosterone plays the role of synchronizer, each evening (peak) it induces the synthesis of tissue specific enzymes in G0 cells and simultaneously inhibits the DNA synthesis in cycling cells. The same parameters have been studied during the different stages of hepatocarcinogenesis induced by Diethylnitrosamine (DEN). After initiation alone, (DEN for 2 weeks) circadian control is unchanged and precancerous cells are not able to reach malignancy. Promotion (DEN for 6 weeks) consists of disturbing the circadian synchronization to liberate the selective growth of initiated precancerous cells. This proliferation advantage favours the accumulation of chromosomal aberrations including those implicated in malignant transformation: i.e. activation of oncogenes or inhibition of antioncogenes.

Nuclear lesions during rat hepatocarcinogenesis. I. Measuring the sister-chromatid exchanges during initiation, promotion and progression of rat hepatocarcinogenesis induced with diethylnitrosamine.

Cytogenetic endpoints such as sister-chromatid exchanges (SCEs), chromosomal aberrations and micronuclei (MNs) have been widely used as indicators of genetic damage. However, no systematic attempts have been made to correlate the levels of these cytogenetic endpoints with the different steps of carcinogenesis. In the present report, the induction, accumulation and persistence of SCEs and high frequency cells (HFCs) were measured in liver cells during the initiation, promotion and progression steps of rat hepatocarcinogenesis induced by diethylnitrosamine (DEN). The results indicate that lesions leading to SCEs accumulate during initiation only. When DEN administration is longer than the duration of this first step, SCE values stabilize. After stopping the carcinogenic treatment, the SCE levels decrease to control values whether or not promotion and progression occur.

Nuclear lesions during rat hepatocarcinogenesis. II. Measuring the micronuclei during initiation, promotion and progression of rat hepatocarcinogenesis induced with diethylnitrosamine.

We reported in our companion paper the strong correlation between elevated sister-chromatid exchange (SCE) frequencies and the initiation step of rat hepatocarcinogenesis. We have also shown that SCEs return to normal values during the promotion and the progression stages. In the present study, we evaluated the clastogenic activity of diethylnitrosamine (DEN) during initiation, promotion and progression of rat hepatocarcinogenesis. We measured, at various times after DEN administration, the number of micronuclei (MN) produced by the mitotic response to partial hepatectomy. The results established that the DEN treatment induces a great number of preclastogenic lesions. In subcarcinogenic conditions (initiation alone), the number of MN expressed after partial hepatectomy remains high regardless of the time interval between the end of the DEN treatment and the operation. In this condition, the preclastogenic lesions persist for up to 1 year after the DEN administration is discontinued. Conversely, in carcinogenic conditions (initiation + promotion + progression), the number of MN expressed after partial hepatectomy decreases during the promotion and progression stages. These observations indicate that promotion and progression but not initiation are associated with the expression of persistent preclastogenic lesions, resulting in the production of chromosomally abnormal hepatocytes.

Strychnopentamine, a potential anticancer agent.

We analysed the effects of strychnopentamine, an alkaloid isolated from Strychnos usambarensis, on an Ehrlich ascites tumor growing in the mouse after inoculation. Four subcutaneous injections of 1.5 mg strychnopentamine (1 per day) induce a significant decrease of the number of tumor cells and a significant increase of the survival of the treated mice. Observed side effects are partial haemolysis and some liver damage.

Cytogenetic changes in hepatocarcinomas from rats treated with chronic exposure to diethylnitrosamine.

Cytogenetic analysis of rat hepatocarcinomas obtained after diethylnitrosamine (DEN) exposure showed a wide variety of numerical and structural chromosomal changes: 53 of 86 hepatocellular carcinomas showed at least one recurrent chromosomal aberration. Some of these recurrent changes occurred in several tumors. Chromosomes 1, 3, 11, and 12 were abnormal in more than 30% of the carcinomas; chromosomes 2, 4, 5, and 10 were abnormal in 10%. Moreover, chromosomes 1 and 10 were generally lost or deleted and chromosome 3, 4, and 11 were very often gained. The most frequent anomaly was loss of chromosome 1 which was observed in 35% of the tetraploid cell populations. The occurrence in several tumors of recurrent chromosomal rearrangements as well as various repeated aneuploidies strongly suggests that these anomalies are implicated in the process of rat hepatocarcinogenesis induced by DEN treatment.

Cell population kinetics and ploidy rate of early focal lesions during hepatocarcinogenesis in the rat.

We have studied the changes in cell population kinetics and DNA-content of cycling parenchymal cells during the very early steps of rat hepatocarcinogenesis in Faber's protocol. Adult rats were initiated by a single dose of diethylnitrosamine (DENA, 200 mg kg-1), followed 2 weeks later by a 2-week diet of 0.03% 2-acetylaminofluorene (2-AAF) as selection phase. In the middle of selection time, a single necrogenic dose of carbon tetrachloride (CCl4, 2 ml kg-1) was administered by gavage. Twenty four hours thereafter, radiolabelled thymidine (3H-TdR, 1.5 microCi g-1) was given by repeated injections during 24 h. An emergence of small, pyroninophilic ('tigroid') foci was observed at the second, fifth and eighth days after the proliferative stimulus. The focal putative precancerous cells presented a significant higher labelling index (L1) than the non-affected parenchymal cells for all exposure times. However, the labelling intensity decreased from the second to the eighth day after CCl4, suggesting a dilution of the radiolabelled DNA by repeated divisions within the foci. The nuclei of the same foci were analysed for DNA-content by feulgen microdensitometry on neighbouring sections. A gradual reduction of nuclear DNA-content was observed in 66% of the foci at the fifth day and in 100% of foci at the eight day, as compared to surrounding tissue and untreated animals, where labelling and DNA-content remain in the same ratio.

Circadian synchronization of liver regeneration in adult rats: the role played by adrenal hormones.

The role played by the adrenal hormones in the regulation of liver proliferation in adult rats was investigated under various experimental conditions. In untreated control groups, cell growth was very low and endogenous corticosterone levels showed a clearly-defined circadian rhythm with a peak in the evening. Adrenalectomy depressed the level of endogenous corticosterone immediately and the growth rate of the liver increased significantly. We were able to prevent this effect by repeated injections of corticosterone at physiological doses. After a 1/3 hepatectomy and a sham-operation, the corticosterone blood level maintained its normal circadian pattern with the exception of a transient increase during the first two post-operative hours. After a hepatectomy of this kind, a negative correlation was found to exist between the adrenal hormone level and the waves of DNA synthesis; the subsequent mitoses appeared in two successive circadian waves of decreasing amplitude, a maximum value being reached in the morning. In rats submitted to a 1/3 hepatectomy and an adrenalectomy simultaneously, the endogenous corticosterone level fell significantly after a post-operative peak. The regenerating pattern was completely different from that induced by 1/3 hepatectomy alone. The rise in the labelling index began earlier and rose to significantly higher values; it was then followed by a single large mitotic wave without any circadian rhythm. These results favour the hypothesis that adrenal hormones have a significant effect on the negative control of liver regeneration. Circadian changes in the corticosterone level were responsible for the nycthemeral pattern observed in the regenerating liver after a partial hepatectomy. The results show a marked inhibition of the G1-S transition, particularly in the evening, when the endogenous corticosterone concentration was at its highest. Also discussed is the relationship between corticoids and 'chalones', which synergetically inhibit the passage from G0 into the cell cycle.

Action of phenobarbital given to rats together with diethylnitrosamine on the O6-ethylguanine content of liver DNA.

When rats are fed diethylnitrosamine (10 mg/kg/day), no O6-ethylguanine is found in liver DNA after 2 weeks, but a considerable amount accumulates after 4 weeks. On the other hand, a 2-week feeding of diethylnitrosamine is not sufficient to induce liver cancers, whereas a 4-week treatment leads to hepatocarcinomas in 50% of the animals. Administration of phenobarbital (75 mg/kg/day) together with diethylnitrosamine during 4 weeks prevents the formation of liver cancers. It also prevents accumulation of O6-ethylguanine in liver DNA. Phenobarbital does not change the amount of O6-ethylguanine repair activity present in liver chromatin after 2 or 4 weeks of treatment with diethylnitrosamine. It is thus concluded that, by inducing the development of the endoplasmic reticulum, phenobarbital decreases the equilibrium concentration of the ultimate carcinogen derived from this indirect alkylating agent.

Circadian synchronization of hepatocyte proliferation in young rats: the role played by adrenal hormones.

The circadian rhythm of hepatic cell proliferation in rats appears on the 20th day of life, when the hypothalamo-adrenal axis is mature enough for circadian activity to occur. From the 20th day to the 30th day of life, the mitotic rhythm is progressively induced by a reduction in nocturnal values, while diurnal rhythms remain unchanged. Mitotic peaks emerge at 10.00 hours. A labelling index wave occurs 8 hr before the corresponding mitotic wave, with a peak at 02.00 hours and a minimum in the evening, coincidental with the acrophase of plasma corticosterone level (activity phase). Labelled mitoses curves and metaphase accumulation after colchicine injection show that the duration of the S, G2 and M phases remain approximately constant and that the circadian variation is due to a variation in the rate of cells that enter these successive phases. During the synchronization period (from day 20 to 30), the growth fraction decreases progressively. Adrenalectomy at this time is followed by a higher cell proliferation and all rhythms disappear after 2 days. Corticosterone injected before the triggering of the rhythmic activity in 17-day-old rats immediately reduces the labelling index, while the mitotic index is decreased 10 hr later; this delay is equal to the S + G2 duration. The results are discussed. They favour the hypothesis that the circadian variation of corticosterone is responsible for the induction of a circadian variation in developmental cell proliferation by inhibition of the G1-S transition when it is higher in the evening.

Anti-carcinogenic action of phenobarbital given simultaneously with diethylnitrosamine in the rat.

The present work has been planned in order to elucidate the effect of phenobarbital (PB: 15 mg per rat of ingested dose) on carcinogenesis when it is administered simultaneously with diethylnitrosamine (DEN: 10 mg/kg/day). Wistar rats (180 g) were treated by DEN alone or by DEN + PB during 2, 4 and 6 weeks according to our schedule for hepatocarcinogenesis. After the end of the treatment, the number and the size of induced PAS positive preneoplastic foci was significantly reduced when PB was given simultaneously with DEN for 4 and 6 weeks. The mitotic inhibition and the production of micronuclei normally observed after partial hepatectomy in DEN treated rats were also significantly decreased in DEN + PB treated rats. When the treatment last only 2 weeks, the presence of PB did not change significantly the last parameters. In DEN + PB treated rats, the survival was prolonged and the tumor incidence decreased as compared with the results obtained by DEN alone. It is concluded that PB, which promotes carcinogenesis when administered after the DEN treatment, reduces the carcinogen effect when given simultaneously with DEN. This 'anti-carcinogen' effect acts on the initiation as well as on the promotion of the precancerous lesions. Biochemical investigations are in progress to obtain more information about this 'paradoxical' PB effect.