Evaluation of the genotoxic potential of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites, glycidol and beta-chlorolactic acid, using the single cell gel/comet assay.

3-monochloropropane-1,2-diol (3-MCPD) is a member of a group of chemicals known as chloropropanols. It is found in many foods and food ingredients as a result of food processing. 3-MCPD is regarded as a rat carcinogen known to induce Leydig-cell and mammary gland tumours in males and kidney tumours in both genders. The aim of our study was to clarify the possible involvement of genotoxic mechanisms in 3-MCPD induced carcinogenicity at the target organ level. For that purpose, we evaluated DNA damages in selected target (kidneys and testes) and non-target (blood leukocytes, liver and bone marrow) male rat organs by the in vivo alkaline single cell gel electrophoresis (comet) assay, 3 and 24 h after 3-MCPD oral administration to Sprague-Dawley and Fisher 344 adult rats. 3-MCPD may be metabolised to a genotoxic intermediate, glycidol, whereas the predominant urinary metabolite in rats following 3-MCPD administration is beta-chlorolactic acid. Therefore, we also studied the DNA damaging effects of 3-MCPD and its metabolites, glycidol and beta-chlorolactic acid, in the in vitro comet assay on CHO cells. Our results show the absence of genotoxic potential of 3-MCPD in vivo in the target as well as in the non-target organs. Glycidol, the epoxide metabolite, induced DNA damages in CHO cells. beta-Chlorolactic acid, the main metabolite of 3-MCPD in rats, was shown to be devoid of DNA-damaging effects in vitro in mammalian cells.

Lack of genotoxicity of potassium iodate in the alkaline comet assay and in the cytokinesis-block micronucleus test. Comparison to potassium bromate.

Iodine could be added to the diet of human population in the form of iodide or iodate but iodate had not been adequately tested for genotoxicity and carcinogenicity. In the present study, genotoxic effects of potassium iodate were evaluated in vitro using the alkaline comet assay and the cytokinesis-block micronucleus assay on CHO cells and compared to halogenate salt analogues potassium bromate and chlorate and also to their respective reduced forms (potassium iodide, bromide and chloride). The results showed that the comet assay failed to detect the presence of DNA damage after a treatment of cells by potassium iodate for concentrations up to 10 mM. This absence of primary DNA damage was confirmed in the cytokinesis-block micronucleus assay. In the same way, results showed that potassium chlorate as well as potassium iodide, bromide and chloride did not induced DNA damage in the alkaline comet assay for doses up to 10 mM. By contrast, potassium bromate exposure led to an increase in both DNA damage and frequency of micronucleated cells. The repair of bromate-induced DNA damage was incomplete 24 h after the end of treatment. These results seem to indicate that potassium bromate would induce DNA damage by several mechanisms besides oxidative stress.

Effects of griseofulvin in medium-term liver carcinogenesis assay and peripheral blood micronucleus test in rat.

Published data have suggested a possible link between the tumor promoting activity and the aneugenic properties of griseofulvin. The present study was conducted to explore this relationship. Griseofulvin was evaluated both for its potential promoting activity in liver carcinogenesis in partially hepatectomized F344 male rats initiated by diethylnitrosamine and for its genotoxic potential in the peripheral blood micronucleus assay. Rats were treated daily with 2,000 mg/kg body weight by oral gavage for 12 weeks in the medium-term carcinogenesis bioassay. GST-P-positive foci (mean number and surface area) and altered cell foci were compared in the liver of rats treated with griseofulvin alone, diethylnitrosamine alone,and griseofulvin in addition to diethylnitrosamine by using immunohistochemical and histopathological evaluation, respectively. This evaluation allowed the conclusion that griseofulvin did not initiate the carcinogenic process but rather had a potential in the liver for tumor promoting activity. Griseofulvin was found to be negative in the rat peripheral blood micronucleus test when given at a daily oral dose of 2,000 mg/kg body weight for at least 3 weeks.

Evaluation of residual and therapeutic doses of tetracycline in the human-flora-associated (HFA) mice model.

In order to evaluate an in vivo model system for assessing the effect of therapeutic and residue levels of tetracycline on human intestinal microflora, tetracycline was administered via drinking water (1, 10, and 100 mg/liter) to human-flora-associated (HFA) male and female mice. The effects of the antibiotic on fecal aerobic and anaerobic populations, selection of bacteria resistant to tetracycline, metabolic parameters of the microflora, and maintenance of the intestinal barrier against exogenous Salmonella (resistance to colonization) were recorded. In both sexes of mice, tetracycline exposure at 10 and 100 mg/liter induced the selection of several resistant bacterial species (Gram-positive anaerobes, Bacteroides fragilis, enterobacteria, and enterococci). This effect was also observed at the lowest dose (1 mg/liter) in female mice and indicates the potential sensitivity of this endpoint for evaluating the microbiological risk of tetracycline residues. The resistance to colonization was impaired at 100 mg/liter, a concentration corresponding to about half of the therapeutic doses in humans and animals. Metabolic parameters of the microflora were not affected by tetracycline at all levels. In this study, the no-observed-effect level (NOEL) of tetracycline on intestinal flora in this study was less than 1 mg of tetracycline per liter of drinking water. This concentration in the mouse corresponds to 0.125 mg of tetracycline per kilogram of body weight per day. Within the constraints of the experimental design employed here, the HFA mice model proved to be acceptable for studying dose-related effects of tetracycline on human intestinal microflora.

Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis.

BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during apoptosis in comparison with standard DNA flow cytometry analysis. METHODS: Staurosporine-induced apoptosis in CHO cells is an adequate model to study a rapid time- and dose-dependent appearance of this process. RESULTS: Nuclear staining with DAPI confirmed the induction of apoptosis with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.

Mutagenicity of malachite green and leucomalachite green in in vitro tests.

The genotoxic potential of the fungicide malachite green (MG) and its reduced derivative leucomalachite green (LMG) was assessed in bacteria and mammalian cells using the standard Salmonella typhimurium/Ames and CHO/HGPRT tests. In vitro potential DNA damaging effects of MG and LMG were tested using the single-cell gel electrophoresis (Comet) assay on CHO cells. Malachite green was found to be extremely cytotoxic to bacteria and mammalian cells. It did not have any mutagenic activity, in any bacterial strains, in the presence or absence of metabolic activation for doses up to 10 microg per plate. In the CHO/HGPRT test, the mutagenic potential of MG could be evaluated only for very low concentrations ranging from 0.001 to 0.05 microg ml(-1) medium. When S9 fraction was added to the medium, the highest tested dose could be increased to 1 microg ml(-1). In these experimental conditions, MG did not increase the number of thioguanine-resistant mutants. Leucomalachite green was less toxic than MG to Salmonella typhimurium and did not have mutagenic activity in the Ames' test for doses up to 2000 microg per plate. It was also less cytotoxic than MG to CHO cells and was tested at doses ranging from 5 to 100 microg ml(-1). Overall results indicated that LMG was not mutagenic in the HGPRT test. In the Comet assay, MG induced DNA damage only at cytotoxic doses. Loss of cell viability was observed for doses of > or = 3 microg ml(-1), with parallel increase in DNA alterations as measured by the tail moment. After metabolic activation, however, DNA damage was observed at doses (15-20 microg ml(-1)) inducing only low cytotoxicity. In this case, the direct genotoxicity of MG metabolites could not be excluded. In the absence or presence of metabolic activation, LMG did not have any effect on cell viability or DNA damage for doses up to 500 microg ml(-1). This study indicates that LMG, which is the main residue found in fish tissues after treatment with MG, did not have any mutagenic or clastogenic effects in the experimental conditions used.

Early detection of staurosporine-induced apoptosis by comet and annexin V assays.

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction.

DNA damaging effects of pesticides measured by the single cell gel electrophoresis assay (comet assay) and the chromosomal aberration test, in CHOK1 cells.

One herbicide (isoproturon), two fungicides (carbendazim and chlorothalonil) and etoposide (an effective antitumor agent used as a positive control), were tested for their ability to induce cytotoxic and genotoxic effects in Chinese Hamster Ovary (CHOK1) cells. Etoposide induced DNA damage detectable both by the alkaline Single Cell Gel Electrophoresis (SCGE) assay and the chromosomal aberration (CA) test in absence of noticeable cytotoxicity. With the SCGE assay, a clear induction of DNA damage was observed for chlorothalonil within a 0.2 to 1 microM concentration range. In the CA test, chlorothalonil gave also positive results, inducing mainly chromosome breaks. In contrast, no DNA damage was observed with the SCGE assay for carbendazim and isoproturon. In the CA test, carbendazim induced only numerical aberrations in the concentration range of 25 microM to 100 microM, and isoproturon did not induce any significant increase in CA. In conclusion, chlorothalonil appears genotoxic in proliferative CHOK1 cells, and as expected, the aneugenic compound, carbendazim, did not induce DNA strand breaks in the SCGE assay.

Healing of muscle trauma after intramuscular injection of antibiotics in sheep: correlations between clinical, macroscopic and microscopic scores.

The present study aimed to predict the resultant healing from early lesions (found days 3 and 10 post injection) caused by the intramuscular injection of veterinary antibiotic formulations. Nineteen marketed drugs were selected in order to screen a wide range of irritation conditions at the injection site. Nineteen ewes were each injected intramuscularly with one of the formulations. Each injection was at a different site, 3 and 10 days prior to slaughter. Fourteen of these ewes also received intramuscular injections at two other sites 21 and 32 days prior to slaughter. The tolerance was monitored by clinical examination of the injection site and by gross and microscopic pathology. Myodegeneration and fibre necrosis were determined histologically. The clinical scores did not correlate with the other findings. Myodegeneration correlated with the size of the lesion on day 3 post-injection and was not found thereafter. Although occasionally found alone, it was generally associated with and surrounded by fibre necrosis. When myodegeneration was the only lesion, regeneration was complete by day 21 and the fibrosis was minimal or absent. Necrosis at day 10 post-injection correlated with necrosis at days 3, 21 and 32 post-injection. Fibrosis became prominent around the necrotic muscles from day 10 post-injection. Healing from necrosis was slow with, in some instances, encapsulated debris still persisting at day 32 post-injection. The tissue irritation index correlated well with myodegeneration and necrose (acute lesions) and fibrose and necrose (older lesions). Thus, after considering a large sample of antibiotic formulations, this study indicated that healing could be predicted from the muscle fibre histopathology at days 3 and 10 post-injection. If myodegeneration was found alone, full recovery within 21 days could be predicted. If fibre necrosis was extensive, the healing involved encapsulating the necrotic tissues and thus resulted in extensive scar formation. The tissue changes explained why the irritation index of the lesions at days 21 and 32 post-injection could be predicted from their irritation index at days 3 and 10 post-injection. Likewise, the size of the lesion at days 21 and 32 post-injection could not be predicted from its size at day 3 post injection.

[Bioavailability of muscle creatine kinase in sheep. Application to the assessment of local tolerance to injectable veterinary formulations]. / Biodisponibilité de la créatine kinase musculaire chez le mouton. Application à l'évaluation de la tolérance locale de formulations vétérinaires injectables.

Pharmacokinetic variables of skeletal muscle creatine kinase were determined in sheep after intravenous and intramuscular administration of the semipurified enzyme. Catheters implanted in the jugular vein were used for both intravenous injections and blood withdrawals. Blood sample collection by vacutainer and hemolysis may in fact have considerable effects on the measurement of creatine kinase activity in plasma. The change in the enzyme activity versus time in the plasma, after intravenous administration (123 +/- 38 U/kg) of creatine kinase, was fitted by a biexponential model. The mean volume of the central compartment (45 +/- 5 mL/kg) was approximately equal to the plasma volume. Plasma half-life and plasma clearance of creatine kinase were 3.7 +/- 1.7 h and 23 +/- 8 mL.kg-1.h-1, respectively. Mean plasma bioavailability of creatine kinase after intramuscular administration (357 +/- 36 U/kg) in both the loins and the gluteal mass was 42%. Maximal plasma activity was observed 4 and 5 h after injection and the half-life of the terminal phase was 7.3 or 8.6 h according to the muscle. The extent of muscle damage after intramuscular administrations of 21 veterinary drug formulations (one product per animal) was estimated from the total creatine kinase activity released in plasma during the 72 h following the injection. Equivalent weights of damaged muscle ranged from 1.4 to 83.3 g according to the irritant potency of the test formulation. Results differed only moderately between the injection sites (right and left gluteal mass) in the same animal. It can be concluded from this study that, in sheep: i) the bioavailability of creatine kinase from different injection sites (gluteal mass and loins) is comparable; and ii) the intra-individual variability in the estimation of muscle damage is moderate. Once validated, this non-invasive approach for local tolerance studies could be of value in assessing and comparing the irritant potency of veterinary drugs and in reducing the number of animals required.

Oral toxicity in weanling and adult rats and in vitro genotoxicity of the veterinary anthelmintic rafoxanide.

The oral toxicity of the veterinary anthelmintic Rafoxanide was evaluated in newborn rats exposed in utero and during lactation. In another group, 3 months of oral treatment started after weaning. Rafoxanide administration to dams (10 mg kg-1) decreased the number of pups per litter, induced high mortality (42%) and delayed the growth of offsprings before weaning. Histopathological examination of the central nervous system revealed vacuolation of the white matter. Vacuolation was particularly severe in the cerebellum of the 14-21-day-old rats. Rafoxanide induced, in some pups (35%), the formation of cataracts. These toxic effects seemed to be reversible and were no longer detectable after a further 3-month administration of Rafoxanide (10 mg kg-1) to rats born of treated dams. In adult rats treated orally for 3 months with 1, 5 or 25 mg kg-1 Rafoxanide, biochemical and haematological tests did not reveal dose-related effects. Rafoxanide had no mutagenic activity in the Ames and CHO/HGPRT tests.

Time course reversibility of liver and blood changes induced in weanling rats after polychlorinated biphenyl exposure during lactation.

Phenoclor DP5 (DP5), a mixture of polychlorinated biphenyls (PCBs), was administered to lactating rat dams, every two days from postnatal days 2 to 20, at 50 mg/kg of body weight. Resulting effects on tissue concentrations of PCBs and on liver lipids, nucleic acids (DNA and RNA), proteins and blood lipids of offspring, at different times after weaning, until postnatal day 100, were studied. On postnatal day 21, DP5 contents found in liver, brain and fat of young rats indicated that a great part of the dam's body stores of PCBs was eliminated into the milk. These accumulations were related to an increase of relative liver weight, a decrease in DNA concentration and a rise in liver RNA, protein and phospholipid levels, indicating an inducing effect of DP5 in weanling rat. Changes in lipid metabolism were associated with increases of liver triacylglycerol and cholesterol and decrease of blood triacylglycerol. Both sexes exhibited an identical responsiveness to PCBs. The biochemical alterations observed in liver and blood at weaning, disappeared with time and were no longer detectable after postnatal day 40 in both sexes. Relative liver weight increase persisted until postnatal day 60 in males and postnatal day 100 in females. Residual aspects of alterations induced before weaning, by exposure via milk, could persist until postnatal day 100. Permanent effects of PCBs, after perinatal exposure could be assessed in later life of rats, when PCBs were almost completely cleared from body tissues.

Effects of perinatal ivermectin exposure on behavioral development of rats.

The antiparasitic agent ivermectin was administered to pregnant rats from days 6 to 20 of gestation and 2 to 20 of lactation at 1, 2 or 4 mg/kg/day. Reproductive performance and behavior of offspring in the preweaning period were observed. Ivermectin had no effect on reproductive performance and dams' growth. The highest dose induced 100% pup mortality. Later on, ivermectin at 4 mg/kg was administered only during gestation. At that dose, the drug induced 22% mortality and affected temporarily cliff avoidance, locomotion, negative geotaxis and swimming development. At 2 mg/kg, the drug induced offspring mortality (31%), retarded growth and delayed eye opening, cliff avoidance and surface righting reflex, negative geotaxis, locomotion and swimming development. Ivermectin at 1 mg/kg had no effect on mortality and growth but cliff avoidance and locomotion were retarded. Data suggest that newborn rats were highly susceptible to the neurotoxic action of ivermectin. Whether its effects were prenatal or by acute intoxication via mother's milk are discussed.

Biochemical changes in liver and plasma of rats after oral administration of edifenphos.

Rats were treated, during 8 weeks, with different doses of edifenphos (0, 5, 10, 20, 40 mg/kg/day) incorporated in the food. Treatments with 20 and 40 mg/kg increased the relative liver weight and hepatic concentrations of phospholipids, microsomal proteins and cytochrome P450. Aminopyrine N-demethylase and aniline hydroxylase activities were increased at the same doses. Hepatic lipids and blood triacylglycerols remained unchanged by the treatment, but blood cholesterol increased significantly (33%) at 40 mg/kg. Plasma cholinesterase activity was slightly depressed in rats treated with the two higher doses. Under these experimental conditions, the dose of edifenphos, inducing no significant difference was 10 mg/kg/day.

[Polychlorocamphane (P.C.C.) effects on the microsomal enzyme activities and fat content of rat liver (author's transl)]. / Effets du polychlorocamphane (P.C.C.) sur l'activité des enzymes microsomales et la composition lipidique du foie de rat.

The rat liver mixed function oxydases are induced by polychlorocamphane incorporated in the food at a dose level of 250 ppm for 30 days. The fat content of liver is enhanced by the treatment, specially that of microsomal phospholipids and whole liver esterified cholesterol and triglycerides. Microsomal acylcoenzymeA-cholesterol-acytransferase activity, expressed in nM/g of liver weight, which catalyses the esterification of free cholesterol, is increased significantly by P.C.C. treatment. The accumulation of triglycerides is inconsistent with an enhanced lipolysis of adipose tissue since the plasma levels of free fatty acids are the same for control and treated rats. These results confirmed the eventual correlation between induction and hepatic steatosis suggested by some authors.

[Adrenal cortex and avitaminosis A: II. Complementary in vivo and in vitro studies on inhibition of corticoidogenesis in moderate deficiency in the rat. III. In vivo criterion for activity: glycemic response to ACTH]. / Corticosurrénale et avitaminose A: II. Compléments a l'étude in vivo et in vitro; modalités d'inhibition de la corticoidogenèse en carence modérée chez le rat. III. Recherche d'un critère d'activité utilisable in vivo; réponse glycémique a l'ACTH

II. We set up new experiments, both in vivo and in vitro, in order to explain the impaired adrenal response to stress, of vitamin A middly deficient, male Wistar rats. Injections of progesterone, 6 mg s.c. or i.p. every other day, did not improve the deficiency curse in our rats. In agreement with these results, we have found that, in vitro, the delta5-3beta hydroxysteroide dehydrogenase is not altered in deficient glands; we confirmed it histochemically. On the other hand, the 11beta and 18 hydroxylase activities are lowered in vitro (and presumably in vivo). When conditions of hyperactivity are established, the conversion of deoxycorticosterone (DOC) to 18 OH-DOC or to corticosterone is reduced and DOC accumulates in the incubation medium. III. When middly deficient intact rats are injected with ACTH i.p. (2UI p. 100 g of body weight), only some of them are found to have normoglycemia or hyperglycemia, 45 or 60 minutes after injection, as the controls do. Many deficient rats become hypoglycemic as the adrenalectomised animals do. We think that the changes in the glycemia after ACTH injection could be used to discriminate between the more or less deficient rats.