Incorporation of labeled 2,4,5,2',4',5'-hexachlorobiphenyl into the nuclear fraction of rat hepatocytes in vivo.

2,4,5,2',4',5'-[14C] Hexachlorobiphenyl (2,4,5-HCB) a slowly metabolized PCB was given to rats by gastric incubation. The hepatocyte nuclei (HCN) were then isolated and treated with specific hydrolytic enzymes in order to separate the nucleic macromolecules, protein RNA and DNA. The results show that 2,4,5-HCB binds in vivo to hepatocyte nuclei. Liver nuclear proteins bind 70% of 2,4,5-HCB and 30% is found in the DNA fraction. No radioactivity was found in the nuclear RNA fraction at this experimental time (16 h).

Effect of vitamin E dietary intake on in vitro activation of aflatoxin B1.

The molecular mechanism of action of vitamin E on mammalian cells remains to be elucidated. In this study, vitamin E dietary intake was assessed for its effects on the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin E dietary intake and aflatoxin B1 (AFB1) genotoxicity measured in vitro. Thus AFB1 induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to effect of vitamin E dietary intake on hepatic microsomal P-450 content and specific activities involved in AFB1 metabolism. Rats were fed ad libitum a diet containing 0, 0.05, 0.5 or 5 IU of alpha-tocopherol for 8 weeks. Modulation of vitamin E level in postmitochondrial and microsomal fractions resulted in nutritional effects. Cytochrome P-450 content was not modified by the level of vitamin E in the diet. The microsomal P-450 activities, P-450 IIB1 and IIIA, were decreased in the deficient group to -35% and -16%, respectively, as compared with control diet (0.05 IU). Diet supplemented with 0.5 IU of vitamin E increased P-450 IIB and IIIA activities (+28% and +37%, respectively) whereas a diet highly supplemented in vitamin E (5 IU) reduced these specific P-450 activities. Lipid peroxidation, estimated by the formation of thiobarbituric acid reactive products, increased in the dietary vitamin E free diet (+20%) and strongly decreased in the supplemented group (-99%). This study establishes that in vivo, dietary vitamin E protects directly membrane against damage induced by lipid peroxidation and indirectly hepatic microsomal monooxygenase activities. However, vitamin E accumulation seems to alter membrane structure and function. The nutritional effect of vitamin E on hepatic microsomal cytochrome P-450 activities modified the AFB1 genotoxicity measured in vitro.

Effect of vitamin A dietary intake on in vitro and in vivo activation of aflatoxin B1.

The mechanism by which vitamin A prevents or delays in chemical carcinogenesis remains unclear. In the present study, we assess the suggestive role of vitamin A in the initiation phase of carcinogenesis. We have conducted a dose-effect relationship between vitamin A dietary intake and aflatoxin B1 (AFB1) genotoxicity measured both in vitro and in vivo. Thus AFB1-induced mutagenesis in Salmonella typhimurium TA98 was investigated and compared to AFB1-induced single-strand breaks (SSBs) in DNA of rat hepatocytes. Rats were fed ad libitum with diet containing 0, 5, 50 or 500 IU of retinyl palmitate for 8 weeks. The AFB1-treated rats were injected i.p. with 1 mg/kg body weight. In the Ames test conditions TA98 back-reversion was negatively correlated with the log of vitamin A concentration in liver S9 fractions from experimental groups. However, the activities of metabolizing enzymes which specifically activate or deactivate AFB1 were found to be significantly decreased in vitamin A-deficient animals and weakly modified in vitamin A-supplemented animals. For in vivo experiments, the DNA elution rate of both AFB1-treated and untreated rats was increased in vitamin A deficiency condition (+79% and +17% respectively) and was reduced with the higher vitamin A dietary level (-44% and -53% respectively). DNA damage measured in vivo showed a significant positive correlation with mutagenic activity measured in the Ames test. These results confirm that the vitamin A status of animals can influence AFB1 genotoxic activity in vitro and indicate that this phenomenon also occurs in vivo. Thus a similar mechanism may be considered for the protective action of vitamin A both in vitro and in vivo. However, this mechanism is unlikely to involve modulation of the microsomal enzyme system responsible for AFB1 metabolism. Therefore a protective mechanism at the cytosolic or nuclear levels may be suggested.

Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers.

Over the past decade, molecular, biochemical and cellular markers have been extensively used in pollution monitoring of aquatic environments. Biochemical markers have been selected among early molecular events occurring in the toxicological mechanisms of main contaminants. This paper assesses the marine environment quality along the Tunisian coasts using a statistical approach. Clams (Ruditapes decussatus) were collected during the four seasons of 2003 on seven different sites from the Tunisian coasts. Oxidative stress was evaluated in gills using catalase activity (Cat), neutral lipids and malonedialdehyde accumulation. Glutathione S-transferase activity is related to the conjugation of organic compounds and was evaluated in both, gills and digestive glands. Acetylcholinesterase activity was evaluated as the biomarker of exposure to organophosphorous, carbamate pesticides and heavy metals. For each biomarker, a discriminatory factor was calculated and a response index allocated. For each site, a global response index was calculated as the sum of the response index of each biomarker. Discriminant analysis shows significant differences between sites and seasons compared with control sample. Faroua (site 1) and Menzel Jemile (site 2) seem to be the less polluted with respect to the other sites for all seasons. Gargour (site 6) shows the highest Multimarker Pollution Index during the four seasons, indicating higher contamination level.

Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends.

A battery of biochemical parameters was used to evaluate the response of mussels to a contaminated coastal environment. A multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites (BIOMAR European programme). This study allows the evaluation of the temporal trends of this scale when applied to selected sites of European Mediterranean coast (BEEP Biological Effects of Environmental Pollution in Marine Coastal Ecosystems: European programme). Acetylcholinesterase activity (AChE) is highly sensitive to organophosphorus and carbamate insecticides and, to some extent, also to heavy metals. Catalase activity (CAT) and lipid oxidation (evaluated as malonedialdehyde) are markers of oxidative stress, glutathione S-transferase (GST) activity is related to conjugation of organic compounds and benzo(a)pyrene hydroxylase activity (BPH) is a marker of effect of certain planar organic compounds (e.g. polycyclic aromatic hydrocarbons, PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a Multimarker Pollution Index (MPI) was calculated as the sum of the response index of each of the five more discriminating biomarkers. As the result of our calculation method, the quality of the coastal environment at each site can be classified according to a five levels scale. Samples collected for five cruises in May 2001, 2002, 2003, and September 2001 and 2002 showed MPI evolutions. The results show that water quality can be classified from class 1 (clean areas in some sites of France, Italy and Spain) to class 4 (high pollution in main harbours). Results of the use of the biomarker scale in WP3 (Work Package Concernant Biomonitoring Programmes in Mediterranean Sea) during the BEEP programme make a strong contribution to the establishment of standardized strategies and methods for internationally agreed protocols for biomarker-based monitoring programmes. In comparison with scale pollution methodology used in the BIOMAR programme, the main contribution of BEEP was (1) to select from discriminatory analysis the biomarkers to be included in calculation of scale pollution; (2) to improve the use of the biomarker index in order to identify the main contaminants by analysis of individual contributions to the MPI; and (3) to apply methodology for temporal trends at sampled sites.

[Protein metabolism in vitamin A deficient rats. I. Protein and DNA turn-over in rat liver]. / Métabolisme protéique chez le rat carencé en vitamine A. I. Etude du renouvellement des protéines et des ARN hépatiques.

The effect of a vitamin A deficient diet on liver proteosynthesis was studied with in vivo and in vitro methods. Weanling rats obtained from vitamin A rstricted female were fed "separate diet". Control animals received vitamin A in protein diet. Deficient rats were used at the weight plateau stage. The RNA, DNA and protein liver content was unaffected by vitamin A deficiency and the same result was found for free amino acid pool. The turn-over rate of protein and RNA of cellular fractions and total liver was studied. (14C) leucine and (3H) uridine was injected intraperitoneally. Animals were killed at 20-40 minutes and 2-4-6-8-10 days after injection. Synthesis rate of liver proteins was the same in both groups. Our results indicate no difference in the average rate of (14C) protein degradation between control and deficient animals. Vitamin A deficiency decrease the turn-over rate of total ribosomal and soluble RNA. Polyribosome and postribosomal supernatant from deficient rats liver were less active for protein synthesis in vitro than corresponding fractions from well nourished rats. The lesion was located in the post ribosomal supernatant fraction by "crossing over" experiments. Polyribosome desimentation analysis on sucrose gradients revealed no difference between the two groupe. The results indicate that vitamin A deficiency decrease cell proliferation without degeneracy. The limitative agent of proteosynthesis is situated at RNA level. However protein synthesis is unaffected in our experimental procedure.

[Protein metabolism in vitamin A deficient rats. II. Protein synthesis in striated muscle]. / Le métabolisme protéique du rat carencé en vitamine A. II. Etude de la protéosynthése dans le muscle strié.

Parameters of skeletal muscle protein synthesis was studied in vivo and in vitro. The experimental design was previously described. Muscle humidity, protein and DNA content are not modified by vitamin A deficiency. RNA and free amino acids are modified. Glutamine, serine and threonine level decrease while phenylalanine and leucine increase in vitamin A deficients. After a single intra-peritoneal injection of (14C) leucine, the incorporation of radioactivity into total ribosomes and postribosomal supernatant protein was studied. The rats were killed 20, 40, 60 minutes and 2,6,10 days after injections. The half time for radioactivity accumulation was four times higher into total and ribosomal protein, two times higher into postribosomal supernatant protein in vitamin A deficient rats than in well-fed rats. The decay of acid soluble radioactivity was similar in the two groups, although protein synthesis was lowered by vitamin A deficiency. It was then supposed that leucine, enters into other metabolic paths. Sedimentation analysis in sucrose gradients revealed a lower proportion of higher ribosomes species in muscle of deficient rat than in normal ones, but ribosomes from deficients were more active for protein synthesis in vitro than ribosomes from well-fed rats. The vitamin A deficiency decrease the muscle protein synthesis and lead to important changes on in vitro and in vivo ribosomal activity.

[Effect of chronic Phenoclor DP6 ingestion of some liver enzymatic activities in rats (author's transl)]. / Intoxication alimentaire par les polychlorobiphenyles (PCB) chez le rat : V- Influence sur l'activité de quelques enzymes hépatiques.

Rats were fed diet containing 100 ppm (wet weight) of Phenoclor DP6 for 4 weeks. The influence of DP6 was studied by determining the activity of hepatic microsomal enzymes namely : aniline hydroxylase, P-nitroanisole O-demethylase, aminopyrine N-demethylase, methylaniline N-demethylase. The cytochrome P450 and cytochrome b5 liver contents were measured. The activities of Na+ K+ and Mg++ATPases and of G6Phosphatase in the liver were also determined. In the liver of treated rats, the results show a large increased activity of microsomal drug metabolizing enzymes, cytochrome b5 and P450 contents and decreased ATPases and G6Pase specific activities. However total liver activities of ATPases and G6Pase were little modified by ingestion of Phenoclor DP6. The spectra given by cytochrome P450-DP6 binding was a typical type I.

[Effect of polychlorinated biphenyls in the rat: I. -- Relation tissue level dose (author's transl)]. / Intoxication alimentaire par les polychlorobiphényles (PCB) chez le rat.

Rats were fed with a diet containing Phenoclor DP6, a French commercial polychlorinated biphenyl (PCB) mixture, for 30 days. Diet PCB levels were 10, 100, 500 and 1000 ppm (wet weight). At 500 ppm, a 50 per cent mortality was found after 8 days, while a 100 per cent mortality was noted after 6 days with 1000 ppm. Our principal findings include: loss of body weight (at major doses), central nervous system stimulation, inflammation of lungs and gastro-intestinal tractus, and fatty degeneration of the hepatic cells. Ingested PCB is found in all organs and tissues tested and was largely stored in liver and muscle. PCB levels in the major tissues are related to dietary doses.

[Effect of polychlorinated biphenyls in the rat: II.--Preliminary report on metabolic alterations (author's transl)]. / Intoxication alimentaire par les polychlorobiphéniles (PCB) chez le rat.

Rats were fed with a diet containing 10 and 100 ppm (wet weight) of Phenoclor DPR for 4 weeks in order to evaluate the toxicity of a French PCB mixture. The influence of PCB was studied by determining the liver weights, the hepatic and muscular proteins, glycogen and humidity content as well as hematocrite, glucose, proteins and lipids in the plasma, and the lipidic contents of the liver and the carcass. There was a significant increase in both liver to body weight ratios and liver lipid and protein content. A large increase in plasma proteins and lipids was observed at 100 ppm. Liver glycogen content and blood glucose were slightly modified by the PCB intoxication. Muscular parameters remain-ded stable. A consistent effect on liver microsomal proteins was noted for all doses studied. At the same diet levels, Phenoclor DP6 determines more métabolic alterations than Aroclor 1260 or 1268.

[Effect of polychlorinated biphenyls in the rat. III - PCB distribution, storage and elimination relation time (author's transl)]. / Intoxication alimentaire par les polychlorobiphényles (PCB) chez le rat. III - Distribution, stockage et élimination des PCB en fonction du temps.

The study of accumulation and elimination of Phenoclor DP6 (a commercial PCB mixture) was carried out in three experiments : 1) Rats were fed diet containing 100 ppm of DP6 and were sacrificed after 1, 3, 8, 15 and 30 days. 2) Rats fed DP6 diet during 15 days were subsequently fed with a control diet and sacrificed 8, 15, 52 and 78 days after removal DP6 diet. 3) Bile was collected from surgically treated rats fed DP6 diet during 15 days. Polychlorobiphenyls were spotted in fat carcasses, liver, brain, kidney, muscle, feces and urine to determine absorption, distribution in the animal organism and the clearence rates in urine and feces. We found a very hight DP6 absorption and retention. The low excretion and metabolisation of DP6 result in low decrease after removal DP6 diet.

[Effect of polychlorinated biphenyls in the rat. IV - Metabolic alterations and residual effects of DP6 dietary exposure (author's transl)]. / Intoxication alimentaire par les polychlorobiphényles (PCB) chez le rat. IV - modification de quelques paramètres métaboliques au cours de l'intoxication et de la désintoxication.

In the first experiment, rats fed diet containing Phenoclor DP6 at a level of 100 ppm (wet weigh) were sacrificed after 1, 3, 8, 15 and 30 days of treatment. Metabolic alterations such as enlarged liver, increased protein and lipid liver contents and hyperlipemia were established within 8 days. The glucid metabolism and muscle metabolic parameters show a low sensibility to DP6 treatment. In the second experiment, rats fed diet containing Phenoclor DP6 (100 ppm) for 15 days were subsequently removed from the experimental diet and placed on control diet. The metabolic alterations described above disappear within 52 days after removal DP6 diet, although the remaining liver PCB stays elevated. These data attest the early effect of DP6 exposure in rats and the reversibility of induced metabolic alterations.

Effect of dairy products on initiation of precursor lesions of colon cancer in rats.

This study reports the modulating effect of some dairy products on initiation of putative preneoplasic lesions in rat colon (aberrant crypts) by 1,2-dimethylhydrazine dihydrochloride. Uninoculated skim milk, skim milk fermented with Bifidobacterium sp Bio (Danone strain 173010), and a suspension of the same lactic acid bacteria were incorporated in the animals' diet. The tested diets significantly reduced the incidence of aberrant crypts compared with the control diet by 51%, 49%, and 61%, respectively. The effects of the diets on cecal pH, hepatic UDP-glucuronyltransferase activity, and cecal microflora enzyme beta-glucuronidase were also studied. There was no significant difference in cecal pH between rats fed experimental diets and control rat. The diet supplemented with the Bifidobacterium strain suspension significantly decreased only the cecal beta-glucuronidase activity. Both enzyme activities were reduced in rats fed fermented skim milk- or uninoculated skim milk-supplemented diets compared with control animals.

[Metabolic activation of benzo(a)pyrene by human amniotic fluid]. / Activation métabolique du benzo(a)pyrène par le liquide amniotique humain.

The in vitro activation of benzo(a)pyrene was studied in amniotic fluid from ten 4-month pregnant women. Benzo(a)pyrene monooxygenase and epoxide hydrolase activities were in the same range in amniotic fluid as in human liver. Glutathione epoxide transferase activity was markedly lower than in hepatocytes. Human amniotic fluid also catalyzed the formation of hydrocarbon metabolites mutagenic to Salmonella typhimurium TA98 (Ames system). Profiles of amniotic fluid aromatic hydrocarbons from non smokers exhibited low benzo(a)pyrene concentration (less than 0.1 ng/ml).