13-week drinking water toxicity study of hydrogen peroxide with 6-week recovery period in catalase-deficient mice.

A GLP OECD guideline study was conducted to evaluate the subchronic toxicity of hydrogen peroxide (HP) when administered continuously in the drinking water of catalase-deficient (C57BL/6N) mice and reversibility of toxic effects. Groups of mice (15/sex/group) received solutions of 0, 100, 300, 1000 or 3000 ppm HP in distilled water for 13 weeks; five/sex/group continued on untreated distilled water for an additional 6 weeks. Animals drinking 3000 ppm HP exhibited depressed water and food consumption and body weight. Females drinking 1000 ppm HP had reduced water consumption with intermittent effects on food consumption, but no body weight effects. HP administration did not produce any mortality, clinical signs, hematological effects or organ weight effects on brain, liver, kidneys, adrenals, testes, heart or spleen. Total protein and globulin were depressed among high dose males. Mild to minimal duodenal mucosal hyperplasia was noted in animals receiving 1000 and 3000 ppm HP and one male receiving 300 ppm for 13 weeks. There were no other histopathological findings. All effects noted during the treatment period, including the duodenal hyperplasia, were reversible during the 6-week recovery period. Females dosed with 300-3000 ppm HP during the treatment period showed increased water consumption during the recovery period. The no-observed-effect level (NOEL), based on duodenal mucosal hyperplasia, is 100 ppm in drinking water or 26 and 37 mg/kg/day HP, respectively, for males and females.

Bioaccumulation and lack of toxicity of octachlorodibenzofuran (OCDF) and octachlorodibenzo-p-dioxin (OCDD) to early-life stages of zebra fish (Brachydanio rerio).

Previous studies with octachlorodibenzo-p-dioxin (OCDD) and octachlorodibenzofuran (OCDF) in juvenile or adult fish exposed via water revealed no toxicity, despite significant bioaccumulation. With 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), the fish early-life stage study has been shown to be the most sensitive test system. Therefore, the effects of OCDD and OCDF on the early-life stages of zebra fish (Brachydanio rerio) were determined during a flow-through test based on a column generator method. No statistically significant effect of OCDD and OCDF on the survival and hatching time of the eggs was found. Furthermore, no effects on survival, weight, general appearance or behaviour of the larvae were observed at the end of the exposure period of 32 days. GC-MS analysis of test solution samples revealed geometric mean measured concentrations of 32 (OCDD) and 34 ng/l (OCDF), respectively. Concentrations in surviving larvae at the end of the study were 61 (OCDD) and 94 (OCDF) micrograms/kg, respectively. These concentrations in zebra fish larvae were several orders of magnitude higher than concentrations in fish collected from the wild. In a review of the available laboratory fish experiments, we found a lack of biomagnification of OCDD and OCDF. We do not expect to find adverse effects of these compounds on the aquatic environment.

Histochemical detection of the in vivo produced cellular aldehydes by means of direct Schiff's reaction in CCl4 intoxicated rat liver.

A histochemical technique for detection of the in vivo induced cellular aldehydes based on the direct Schiff's reaction is reported in this paper. CCl4-intoxicated rat liver was used as an experimental model. Fresh and non-pretreated rat liver cryostat sections fixed in 10% formol calcium solution and washed in distilled water were exposed to Schiff's reagent. The sections were then immersed in two baths of sodium bisulphite solution, then in water, dehydrated in ethanol, cleared in xylene and mounted in a synthetic anhydrous mounting medium. As Schiff positive areas presented well circumscribed foci which increased with time following intoxication, semi-quantitative planimetric measurements were feasible. The direct Schiff's reaction detects cellular aldehydes in a sensitive, rapid, histologically and topographically estimable way. The appearance of these aldehydes precedes distinctly morphological alterations detectable by other histochemical or histological techniques. No positive results were obtained in control, non-intoxicated rat livers. Inhibition of this direct Schiff's reaction was obtained in positive control rat liver sections preincubated in solutions of aldehyde blockers. Histochemical detection of aldehydes may give useful information on different aspects of tissue and organ intoxication such as their topography, appearance, evolution, extension, consequences and effects of treatment. The direct Schiff's reaction can be considered as a valuable tool in fundamental and applied research dealing with various toxicological, environmental, pathological, cancer-related and therapeutic problems.

Non-toxic potentiation of cancer chemotherapy by combined C and K3 vitamin pre-treatment.

The influence on the survival of ascitic liver tumor (TLT)-bearing mice of combined vitamins C and K3 administered before or after a single i.p. dose of 6 different cytotoxic drugs, all commonly used in human cancer therapy, was investigated. Combined i.p. administration of these vitamins produced a distinct chemotherapy-potentiating effect for all drugs examined, especially when injected before chemotherapy. This potentiating treatment did not increase the general and organ toxicity that accompanies cancer chemotherapy. The possible generation of peroxides followed by membrane lipid alteration, DNase activation and DNA destruction by combined vitamin C and K3 in catalase-deficient cancer cells might be involved in the mechanisms of this selective potentiation.

Changes in nucleolar transcriptional activity and nuclear DNA content during the first steps of rat hepatocarcinogenesis.

Two complementary genetic parameters were followed in liver parenchymal cells during the first steps of rat hepatocarcinogenesis: the expression of nucleolar genes estimated from their silver stainability and the nuclear DNA content determined after Feulgen staining. Putative preneoplastic lesions as foci and nodules were induced by the triphasic 'Gerlans protocol'. Initiation with a single dose of diethylnitrosamine (DEN) was followed by a selection of initiated cells with 2-acetylaminofluorene (2-AAF) in combination with a single necrogenic dose of CCl4 as a proliferative stimulus. Finally after 1 week of normal diet, the animals were treated or not with phenobarbital (PB) for periods up to 2 months. Serial sections were analysed after silver staining (AgNO3), methyl-green--pyronin staining (Unna-Brachet) and Feulgen staining with densitometric and morphometric methods. Silver staining, which is known to stain an acidic protein associated with rRNA synthesis, increased gradually with the duration of the PB treatment. Morphometry revealed an increase in both nucleolar and nuclear volume; the fraction of nuclei with one nucleolus also increased. These results seem to point towards an increase of nucleolar activity in the early steps of PB promotion. Moreover, this shift cannot be ascribed to an increase of DNA content. Indeed, a parallel study on neighbouring sections stained with Feulgen revealed a shift towards a population of diploid nuclei, in contrast to normal liver cells, which are mostly tetraploid. The observed diploidisation may therefore provide a functional advantage for the expansion of putative preneoplastic cells.

Dietary modulation of rat liver carcinogenesis.

Rat liver carcinogenesis was induced according to the resistant hepatocyte model of Solt and Farber. One week after the end of the procedure for the rapid growth of altered hepatocytes, one group of rats was submitted to a high fat (20%) regimen up to the end of the experiment. The incidence of histologically confirmed malignant hepatocarcinomas was compared with that observed in a group that remained on a normal diet. The modulating (promoting) effect of the high fat regimen was evident since nine out of 10 animals in this group bore macroscopically detectable tumors and eight out of 10 presented histologically confirmed hepatocellular carcinomas as early as 24 weeks after the beginning of the experiment. At that time, no malignant tumors were detected in the group submitted to the normal fat regimen. These results are similar to those resulting from a porto-caval shunt or the chronic administration of liver tumor promoters. This suggests that at this stage of the carcinogenic process, any treatment inducing chronically metabolic adaptation in a tissue containing preneoplastic nodules modulates positively the progression of these lesions as demonstrated by the dramatic reduction of the lag period for their malignant transformation.

Promoting effect of oxazepam in rat hepatocarcinogenesis.

In order to check whether the benzodiazepine, oxazepam (OZ), has a modulating effect on the development of liver cancer, it was given to rats previously submitted to two different protocols of hepatocarcinogenesis: the resistant hepatocyte protocol and Pitot's model (initiation-promotion). Its effects were compared with those of phenobarbital (PB) administered under the same conditions. As compared with basal diet, a diet containing 0.05% of PB and 0.1% of OZ enhanced, in both models, the development of gamma-glutamyltransferase-positive lesions in early stages. OZ also had a promoting effect on the development of liver cancers in later stages in both models whereas PB only enhanced it in the resistant hepatocyte protocol. Thus, like PB, OZ may have a promoting effect on liver cancer in rats.

Variations in serum alkaline DNase activity: a possible clinical test for therapeutic prognosis of human tumors.

Previously published histochemical observations indicated that variations in serum alkaline DNase activity (SADA) could be considered as a possible prognostic test for human tumor therapy. In more than 80 cancer patients biochemical measurements of SADA were performed using the spectrophotometrical technique. A decrease of SADA promptly after the beginning of tumor treatment (phase I) may be interpreted as an early sign of therapeutically induced tumor necrosis and as a positive response to the treatment. A delayed regain of SADA (phase II) can predict the long term evolution of the disease. In this phase (II), a regain of SADA up to values higher than the initial value corresponds to a complete tumor regression. If the regain is limited to values lower than the initial value, only a partial tumor regression is seen. No variations of SADA were observed in patients without therapeutic response and with fatal evolution.

Comparison of the biological effects of phenobarbital and nafenopin on rat hepatocarcinogenesis.

In order to further analyze the biological effects of phenobarbital (PB) and nafenopin (NAF) on rat hepatocarcinogenesis, four experiments were undertaken. In the first one, their "promoting" effect on an ongoing carcinogenic process was analyzed. Rats were initiated by diethylnitrosamine treatment (I) and submitted two weeks later to a selection procedure (S). One week after 2-acetylaminofluorene (2-AAF) release, the animals received for up to 56 weeks a basal diet or a diet containing 0.05% of PB or 0.1% of NAF. The quantitative analysis of the gamma-glutamyl-transferase-positive lesions showed that, 8 to 19 weeks after I, PB enhanced the development of preneoplastic lesions whereas NAF inhibited it as compared to a group receiving a basal diet. However, both compounds enhanced the incidence and the yield of liver cancer starting 27 weeks after I (67% and 95%, respectively, vs 10%). In the second experiment, the effect of chronic administration of PB and NAF given after I without S or after S without I was analyzed. Within a period of observation of 27 to 32 weeks, the incidence of cancer was 10% after I/PB and 75% after I/NAF. No cancer developed after S/PB, S/NAF or NAF alone. The third experiment was designed to test whether NAF had an initiating or selecting effect. The results of the quantitative analysis of the gamma-glutamyl-transferase-positive lesions showed that as compared to diethylnitrosamine, NAF had no initiating effect. When NAF replaced 2-AAF in the selection procedure, few gamma-glutamyl-transferase-positive lesions and no cancer were detected 8 and 32 weeks after I. The fourth experiment indicated that NAF could not prevent the remodeling of preneoplastic lesions induced in the I/S protocol. Even though they both have a "promoting" effect in liver carcinogenesis as evidenced by the increased incidence and yield of cancer, PB and NAF act differently.

Comparative analysis of the effect of phenobarbital, dichlorodiphenyltrichloroethane, butylated hydroxytoluene and nafenopin on rat hepatocarcinogenesis.

In order to investigate whether different 'promoters' have the same qualitative and/or quantitative effects on rat hepatocarcinogenesis, 0.05% of phenobarbital (PB), 0.05% of dichlorodiphenyltrichloroethane (DDT), 0.5% butylated hydroxytoluene (BHT) and 0.1% of nafenopin (NAF) were chronically administered in the diet to rats previously submitted to an initiation by diethylnitrosamine and a selection with 2-acetylaminofluorene plus CC14. The animals were killed after 3, 6 and 14 weeks of 'promoters' administration to analyse their effect on premalignant lesions. The quantitative analysis of the gamma-glutamyltransferase positive lesions indicates that as compared to a control group receiving a basal diet after initiation and selection, PB, DDT and BHT enhance the development of these lesions whereas NAF inhibits it. Rats were also killed after 22 weeks of administration to analyse the incidence and the yield of liver cancer. As compared to the control group, PB, DDT and surprisingly NAF enhance the development of liver cancer whereas BHT does not. This suggests that the effect of potential 'promoters' should be analysed on cancer development rather than on premalignant lesions.

Influence of the nature and the dose of the initiator on the development of premalignant and malignant lesions in rat hepatocarcinogenesis.

Using a triphasic protocol recently described to induce malignant tumors in rat liver, the question has been asked whether both the nature and the dose of the initiator influence the carcinogenic process. Two nitrosamines (diethylnitrosamine DEN and N-nitrosomorpholine NNM) have been used to initiate that process. With regard to the premalignant stages appearing in the liver up to 19 weeks after initiation, there is a dose-dependent relationship between the dose of initiator and both the percentage of the parenchyma occupied by and the number of GGT+ lesions. DEN is always more potent than equivalent doses of NNM. With regard to malignant tumors appearing within the period of observation (up to 44 weeks), the two highest doses (100 and 200 mg/kg) of DEN appear to be the only carcinogenic treatments. Cancer incidence (percentage of rats bearing histologically characterized malignant tumor) is the same after both treatments, but the tumor yield (number of tumors per rat bearing macroscopic tumors) is higher (+/- 2X) after 200 mg/kg than after 100 mg/kg.

Increased silver stainability of metaphase chromosomes from rat hepatocytes during in vivo hepatocarcinogenesis.

Silver stainability of metaphase chromosomes was studied in hepatocytes obtained from rats exposed or not to a partial or complete carcinogenic treatment with diethylnitrosamine and phenobarbital. An increased hyperstaining is reported in the carcinogen-treated animals.

Nucleolar changes during the first steps of experimental hepatocarcinogenesis in rats.

Silver stainability of hepatocytes as an expression of nucleolar activity was studied in vivo during rat hepatocarcinogenesis. Male Wistar rats were injected with one dose of diethylnitrosamine (200 mg/kg body weight dissolved in 0.9% NaCl), followed by a selection procedure with a short exposure to 2-acetylaminofluorene in combination with a proliferative stimulus, such as the administration of CCl4. Finally, after 1 week of a normal diet, some of the rats were treated with phenobarbital. After enzymatic isolation, the hepatocytes were silver stained; the estimation of nucleolar activity was determined by a cytomorphologic analysis of the silver-stained nuclei. It was demonstrated that during the first steps of hepatocarcinogenesis, both diethylnitrosamine, as initiator, and phenobarbital, as promotor, induce modifications of the nucleolar morphology in silver-stained hepatocytes.

Promoting effect of portocaval anastomosis in rat hepatocarcinogenesis.

The effect of a surgical intervention, portocaval anastomosis (PCA) has been investigated in three different phases of a triphasic protocol of rat hepatocarcinogenesis. This protocol consists of the i.p. injection of 200 mg/kg of diethylnitrosamine (initiation) followed 2 weeks later by a 2 weeks diet with 2-acetylaminofluorene (2-AAF) and in the middle a necrogenic dose of CC14 (selection). One week later, a promoter such as phenobarbital (PB) is given chronically (promotion). PCA or a sham operation is performed 5 months before the end of the experiment. PCA alone does not induce hyperplastic nodules, nor does it when rats are initiated 5 weeks before. However, PCA alters the evolution of established nodules: it can act as a promoter like PB. Indeed, 5 months after initiation and selection, the number of rats bearing hepatocarcinomas is zero out of seven with a sham operation and six out of seven with PCA performed one week after the end of the selection. The combination of PCA and PB is more potent than PB or PCB alone since six out of six, five out of eight and six out of seven rats, respectively, bear malignant liver tumours. Thus PCA, which induces many systemic perturbations, has a promoting effect. This suggests that the chronic administration of a xenobiotic is not the only way to promote cancer development.

[Comparative systemic analysis of experimental models of chemical hepatic carcinogenesis in the rat]. / Analyse systémique comparative de modèles expérimentaux de cancérogenèse hépatique induite chimiquement chez le rat.

The aim of this work is to compare several experimental protocols inducing hepatocarcinogenesis in the rat. The results show that the experimental protocols using an initiator and a promotor are not equivalent (in term of latency period and premalignant lesions) to experimental protocols using a chronic administration of one carcinogenic compound.

[Promoting effect of portacaval anastomosis in experimental hepatic carcinogenesis]. / Effet promoteur de l'anastomose portocave dans l'hépatocarcinogenèse expérimentale.

A promoting effect of portocaval anastomosis in a triphasic model of rat hepatocarcinogenesis is demonstrated since after initiation and selection premalignant lesions and hepatocellular carcinomas increase. This demonstrates that chronic administration of an exogenous compound is not the only way to promote cancer development.

Comparison of different models of rat liver carcinogenesis: conclusions from a systemic analysis.

Different protocols of chemically induced hepatocarcinogenesis were applied to Wistar rats under identical experimental conditions. The following conclusions may be drawn after an analytic comparison of these results. Various chemical carcinogens show different carcinogenic capacities. Diethylnitrosamine is more potent than N-nitrosomorpholine which is more active than 2-acetylaminofluorene. A short-term exposure to such carcinogens is sufficient to initiate but not necessarily to complete the carcinogenic process. It can be promoted to completion by either a noncarcinogenic promoter or a carcinogen. From a systemic point of view, it appears that, as in the skin models, two-step protocols are not always equivalent to protocols using the same agent during the whole treatment. Moreover, the results observed with a multistep protocol indicate that during the initiating phase the carcinogen plays a selective role distinct both from a pure initiating role and from the promoting effect. The results obtained lead to the conclusion that the distinction between initiation and promotion remains purely operational as it still does not correspond to the nature of well-established biologic processes.

Phenobarbital as a promoter in the initiation/selection process of experimental rat hepatocarcinogenesis.

Supplementary introduction of a phenobarbital (PB) promotion step after the Solt and Farber procedure dramatically increases the number of phenotypically-altered hepatocytes. These hepatocytes occupy approximately 40% of the liver volume after one week of PB treatment. These areas constitute a relatively uniform cellular population with altered histological phenotype and with distinct histochemical markers used by other authors for the detection of premalignancy. This procedure leads to the appearance of numerous hepatocellular carcinomas at approximately the 36 weeks stage. It was suggested that the early hepatocellular alterations after the initiation/selection procedure followed by PB might correspond to the hyperplasia of phenotypically-altered epidermal cells at the conversion step of mouse skin tumor promotion.

Morphological alterations and DNase deficiency in phenobarbital promotion of N-nitrosomorpholine initiated rat hepatocarcinogenesis.

Tumorigenic effect in rat liver was increased when phenobarbital was given chronically after N-nitrosomorpholine. In these rats the liver parenchyma surrounding pre- and neo-plastic lesions demonstrated distinct, mainly centrilobular zones of hypertrophic hepatocytes with abundant eosinophilic, filamentous cytoplasm, increase in nucleic acids and decrease in DNase activity. These alterations might be considered as signs of tumor-promoting activity of phenobarbital.