Genotoxicity in rats treated with the antidiabetic agent, rosiglitazone

Rosiglitazone (RSG), a member of the thiazolidinedione class of antidiabetic agents, improves glycemic control by increasing insulin sensitivity. The therapeutic mode of action of RSG involves its activity as a highly selective and potent agonist for peroxisome proliferator-activated receptor-gamma. Although other drugs in this class have displayed unacceptable hepatotoxicity, RSG was approved for human use. The package insert indicates that RSG has minimal genotoxicity, but information on the genotoxicity of RSG is not available in the published literature. In this study, we used the single cell gel electrophoresis (SCGE)/Comet assay to investigate the DNA damage in peripheral blood and liver cells of rats treated with RSG. Sixteen male Sprague-Dawley rats were randomly distributed into four groups, and dosed daily by oral gavage with 0.0, 0.5, 1.0, and 2.0 mg/kg/day RSG. The rats dosed with 2.0 mg/kg/day RSG received an approximately 10-times the area under the curve concentration of the maximum recommended human daily dose. After 14 days of treatment, the rats were euthanized, and peripheral blood and liver were collected and processed for the Comet assay. A dose-dependent increase in DNA damage (as assessed by % tail DNA and Olive Tail Moment) was observed in the hepatocytes of RSG-treated groups, with significant increases detected between rats treated with all the doses of RSG and the control, and between rats treated with different RSG doses (P < 0.05 - P < 0.0001). In contrast, DNA damage was detected in peripheral blood lymphocytes only in rats treated with the higher RSG doses (1.0 and 2 mg/kg/day). Taken together, the data indicate that RSG is able to induce primary DNA damage in rats, with greater damage being detected in liver cells than lymphocytes.     

Spinal cholinergic involvement after treatment with aspirin and paracetamol in rats

Aspirin and paracetamol have been shown to suppress non-inflammatory pain conditions like thermal, visceral and mechanical pain in mice and rats. The non-inflammatory antinociception appears to be mediated by central receptor mechanisms, such as the cholinergic system. In this study, we tested the hypothesis that the non-inflammatory antinociception of aspirin and paracetamol could be mediated by an increase of intraspinal acetylcholine release. Microdialysis probes were placed intraspinally in anesthetized rats for acetylcholine sampling. Subcutaneously administered aspirin 100 and 300 mg/kg increased, while paracetamol 300 mg/kg decreased intraspinal acetylcholine release. Intraspinal drug administration did not affect acetylcholine release. Our results suggest that an increased intraspinal acetylcholine release could be involved in part of the non-inflammatory pain suppression by aspirin, but not by paracetamol.     

Genotoxicity and cytotoxicity in multiple organs induced by titanium miniplates in Wistar rats

Internal fixture materials are currently used as metallic biomaterials in rehabilitation of human body. Nevertheless, metal release due to corrosion phenomena appears to play a crucial role for human health. Thus, the goal of the present study was to evaluate whether liver, kidney, and lung are particularly sensitive organs for DNA damaging and cytotoxicity following implantation of internal fixture materials composed by titanium alloy in vivo. A total of 18 rats underwent surgical titanium miniplates in their tibias, being randomly distributed into three groups: 30 days, 90 days, and 180 days after implantation. A total of six animals served as negative control (animals that not received any implant). After experimental design, the lung, liver, and kidney were removed for histopatological and genotoxic analysis as depicted by H.E. stain and single cell gel (comet) assay, respectively. No significant statistically differences (p > 0.05) for DNA damaging were found to all experimental groups when compared to negative control for all organs evaluated. In addition, no remarkable morphological alterations were detected under histopathological analysis. Taken together, such results suggest that titanium miniplates are neither able to induce DNA damage in multiple organs nor to cause some abnormalities in lung, liver, and kidney.     

Effect of acute and repeated administration of paracetamol on opioidergic and serotonergic systems in rats

Objective and design: We investigated the antinociceptive effect of paracetamol or morphine after repeated administration and the changes in the characteristics of central μ-, κ- and 5-HT₂ receptors. Treatment: Male rats were injected twice a day for seven days with paracetamol (400 mg/kg, i. p.) or morphine (5 mg/kg, s. c.). Methods: The antinociceptive effect was evaluated 30 min after single and multiple doses of paracetamol and morphine through the hot-plate test. Binding techniques were used to evaluate the receptor characteristics in the frontal cortex. Results: Both paracetamol and morphine induced an antinociceptive effect on day 1 but only paracetamol maintained this effect for seven days while morphine did not. The number of μ-opioid receptors decreased on days 1, 3, and 7 by a similar percentage after paracetamol administration (by 29, 31 and 34 %, respectively), while morphine produced a progressive decrease in comparison with controls (by 37, 49 and 60 %, respectively) and κ-opioid receptors were unaffected. Both drugs similarly decreased the 5-HT₂ receptor number on all days of treatment (by about 30 %). Conclusions: The opioidergic and serotonergic systems are involved in different ways in the induction and maintenance of antinociception after paracetamol or morphine treatment. Received 10 May 2006; returned for revision 14 September 2006; accepted by G. Geisslinger 30 October 2006     

The effects of royal jelly on liver damage induced by paracetamol in mice

The present study was undertaken to investigate the protective effect of royal jelly against paracetamol-induced liver damage. The study was conducted in 90 female Swiss Albino mice, and six groups were established. While the first group was maintained as control, Groups 2–6 were administered 200 mg/kg RJ for 1 day, 200 mg/kg RJ for 7 days, 400 mg/kg PAR for 1 day, 200 mg/kg RJ plus 400 mg/kg PAR for 1 day and 200 mg/kg RJ for 7 days and then second 400 mg/kg PAR on the 7th day, orally, respectively. It was shown that PAR significantly increased serum ALT, AST, ALP, liver MDA levels and significantly decreased liver GSH-Px activity, when compared to the control group (Group 1). On the other hand, meaningful changes were observed in the biochemical parameters of the group which was administered long-term RJ (Group 6). The aforementioned parameters which were statistically significant were determined to have drawn closer to values of the control group, and among these, the existing statistical differences for MDA level and GSH-Px activity between the trial group (Group 6) and the control group disappeared (Group 1). Compared to the pathological changes observed in the liver parenchyma, remark cords, sinusoids and hepatocytes in the group which was administered paracetamol alone (Group 4), lesions were determined to be less severe particularly in the group (Group 6) which received royal jelly for 7 days prior to paracetamol. In conclusion, the administration of royal jelly as a hepatoprotective agent for 7 days against paracetamol-induced liver damage was determined to exhibit marked protective effect on liver tissue.     

Genotoxicity studies on urine and bone marrow samples of rats bearing transplanted nephroma

It was demonstrated earlier that urine of rats bearing transplantedmesoblastic nephroma had high mutagenic activity in Salmonellatyphimurium, which could not be detected in serum samples ofthe same animals. In this paper, cytogenetic alterations arediscussed and the lack of enhanced micronucleus formation inbone marrow of tumorous rats is described. The cytogenetic effectof the hydrophobic (XAD-4) urinary fraction, which has beenfound to be mutagenic in the TA98 Salmonella strain, was examinedin CBA mice. Sister chromatid exchange (SCE) analyses were performedon bone marrow cells of animals treated with single injectionsof concentrated urine samples. Significant and continuous increasescould be detected in the SCE frequencies caused by the urinaryconcentrates with development of the tumour. Pseudouridine,a suggested urinary tumour marker nucleoside, was also studiedfor mutagenicity in the Ames Salmonella test. Both derivatives( and ß), however, failed to induce mutations in theTA98/TA100 strains, either with or without metabolic activation.In conclusion, urinary mutagen(s) produced during the renaltumour growth have a spectrum of genotoxicity involving at leasttwo endpoints, but the high pseudouridine excretion may notbe responsible for these effects.     

Genotoxicity induced in CD-1 mice by inhaled lead: differential organ response.

Lead is perhaps the longest used and best recognized toxic environmental chemical and it is still being used recklessly. Lead (Pb) has been found to be capable of eliciting a positive response in an extraordinarily wide range of biological and biochemical tests; among them tests for enzyme inhibition, fidelity of DNA synthesis, mutation, chromosomal aberrations, cancer and birth defects. Since inhalation is one of the most important routes of environmental Pb exposure, in the present study a lead inhalation model in mice was implemented in order to detect the induction of genotoxic damage as single-strand breaks and alkali-labile sites in several mouse organs (nasal epithelial cells, lung, whole blood, liver, kidney, bone marrow, brain and testes), assessed by single cell gel electrophoresis (SCGE) or Comet assay. We found differences among the organs studied after a single and subsequent inhalations: in the organs analyzed we observed a positive induction of DNA damage after a single inhalation only in the liver and the lung. In subsequent inhalations the response was positive in all organs except the testicle, however, DNA damage induction over time was different for each organ. A correlation between length of exposure, DNA damage and metal tissue concentration was observed for lung, liver and kidney. Differences in DNA damage occurred in organs when lead acetate was administered acutely or sub-chronically. These results show that lead acetate inhalation induces systemic DNA damage but that some organs are special targets of this metal, such as lung and liver, depending in part on length of exposure, suggesting alternative organ processes to handle lead intoxication.     

Genotoxicity of acrylamide and its metabolite glycidamide administered in drinking water to male and female Big Blue mice

The recent discovery of acrylamide (AA), a probable human carcinogen, in a variety of fried and baked starchy foods has drawn attention to its genotoxicity and carcinogenicity. Evidence suggests that glycidamide (GA), the epoxide metabolite of AA, is responsible for the genotoxic effects of AA. To investigate the in vivo genotoxicity of AA, groups of male and female Big Blue (BB) mice were administered 0, 100, or 500 mg/l of AA or equimolar doses of GA, in drinking water, for 3-4 weeks. Micronucleated reticulocytes (MN-RETs) were assessed in peripheral blood within 24 hr of the last treatment, and lymphocyte Hprt and liver cII mutagenesis assays were conducted 21 days following the last treatment. Further, the types of cII mutations induced by AA and GA in the liver were determined by sequence analysis. The frequency of MN-RETs was increased 1.7-3.3-fold in males treated with the high doses of AA and GA (P < or = 0.05; control frequency = 0.28%). Both doses of AA and GA produced increased lymphocyte Hprt mutant frequencies (MFs), with the high doses producing responses 16-25-fold higher than that of the respective control (P < or = 0.01; control MFs = 1.5 +/- 0.3 x 10(-6) and 2.2 +/- 0.5 x 10(-6) in females and males, respectively). Also, the high doses of AA and GA produced significant 2-2.5-fold increases in liver cII MFs (P < or = 0.05; control MFs = 26.5 +/- 3.1 x 10(-6) and 28.4 +/- 4.5 x 10(-6)). Molecular analysis of the mutants indicated that AA and GA produced similar mutation spectra and that these spectra were significantly different from that of control mutants (P < or = 0.001). The predominant types of mutations in the liver cII gene from AA- and GA-treated mice were G:C-->T:A transversions and -1/+1 frameshifts in a homopolymeric run of Gs. The results indicate that both AA and GA are genotoxic in mice. The MFs and types of mutations induced by AA and GA in the liver are consistent with AA exerting its genotoxicity in BB mice via metabolism to GA.     

Parathymic lymph nodes in rats and mice

A number of lymph nodes lie in close proximity to the thymus of mice and rats. In mice, the parathymic nodes are found in the capsule, whereas in rats, the nodes lie on the capsule. The total weight of parathymic nodes in any one animal is one-tenth to one-twentieth that of the thymus.     

Mucolytic activity of azelastine in mice and rats

The mucolytic activity of azelastine, an antiallergic/antiasthmatic drug, in mice and rats was investigated. The oral administration of test compounds 30 min before phenol red i.p. injection stimulated dye secretion in the trachea of mice. The resulting oral ED₅₀'s (mg/kg) were: azelastine, 0.16; salbutamol, 2.5;N-acetylcysteine, 61.8;S-Carboxymethyl-l-cysteine, <100; bromhexine, >100; and potassium iodide, 200. In rats, several drugs stimulated secretion of fluorescein sodium (FINa) in the tracheobronchial lumen. The resulting oral ED₅₀'s (mg/kg) were: azelastine, 0.33; terbutaline, 0.3; salbutamol, 0.89; andS-carboxymethyl-l-cysteine, 56.8. Terfenadine and diphenhydramine (1–10 mg/kg, p.o.) did not stimulate tracheal secretion in rats and mice. The mucolytic activity of azelastine may contribute to its overall effectiveness, including antitussive activity in asthmatics. Finally, this activity seems to be dissociated from its H₁-histamine receptor blocking activity.     

Polyamine metabolism in muscles of mice and rats

Polyamine biosynthesis in different types of muscle was studied in mice and rats. A sex difference of polyamine biosynthesis in the gastrocnemius of the mouse was demonstrated. Ornithine decarboxylase activity was found to be several-fold higher in the gastrocnemius of the male mouse than in that of the female. Orchiectomy resulted in a decline of enzyme activity in the gastrocnemius. This effect was reversed by the administration of testosterone. The elevation of ornithine decarboxylase activity in the gastrocnemius by testosterone was reflected in an increased content of the polyamines in the muscle. Muscles of other types, i.e. soleus, heart and urinary bladder were shown to be virtually unresponsive to testosterone treatment. Neither were the muscles of the rat, including gastrocnemius, found to be affected by the androgen.     

Genotoxicity of azidoalanine in mammalian cells

Sodium azide mutagenesis is mediated through a metabolic intermediate in bacteria and plant species. However, very little is known about the interaction of this intermediate with nucleic acids, its genotoxic potential, or its mechanism of action, especially in mammalian cells. Chinese hamster cells and normal human skin fibroblasts were treated with extracts from Salmonella typhimurium or Hordeum vulgare (barley) containing a crude mutagenic metabolite, as well as with synthetically produced azidoalanine. The cells were evaluated for the induction of sister chromatid exchanges and the ability to perform unscheduled DNA synthesis. With the purified azidoalanine and the azide-treated extracts from Hordeum vulgare, there was a statistically significant increase in the frequency of sister chromatid exchanges observed in both Chinese hamster cells and human fibroblasts. This increase was about twofold, as compared with the control. On the other hand, there was no detectable genotoxic response when cells were exposed to azide-treated extract from Salmonella typhimurium. The results imply that azidoalanine and the crude mutagenic metabolite from Hordeum vulgare are weakly genotoxic in mammalian cells.     

Lysophosphatidylserine as histamine releaser in mice and rats

Lysophosphatidylserine is a specific inducer of histamine release in isolated mast cells. To determine whether a similar effect is manifestin vivo, the phospholipid was injected (1–5 mg/kg i.v.) into mice and rats. A dosedependent rise in blood histamine was observed in both animals. The several-fold increase in blood histamine occurred in the first minutes and was followed by a slower decline toward normal values. A second dose of lysophosphatidylserine was without effect. Systemic manifestations (depression, hypothermia, hypotension) were associated with the increased blood histamine level. When the tissue histamine stores accessible to lysophosphatidylserine were previously decreased by repeated phospholipid injections, no systemic symptoms occurred. Mobilization of carbohydrate reserves was also manifest during the action of lysophosphatidylserine. Prior treatment with compound 48/80 induced sustained refractoriness to lysophosphatidylserine. Structure-activity relationship demonstrated that the property to induce histamine release was linked to the structure of serine head group. Thus, other natural phospholipids or lysophospholipids were inactive. It is concluded that in analogy with the effect seenin vitro lysophosphatidylserine producesin vivo release of mast cell histamine.     

Mouse IgE-mediated paw anaphylaxis in mice and rats

Anaphylactic swelling is induced in the hind paws of mice and rats by intravenous challenge of antigen 72 h after subplantar injection of mouse IgE-rich antiserum into the paws, and can be quantified rapidly and accurately by measuring paw thickness. The edema model described may serve as an alternative to passive cutaneous anaphylaxis for studying immediate allergic reactions with mouse homocytotropic antibodies and screening potential antiallergic drugs.     

Genotoxicity and cytotoxicity in male B6C3F1 mice following exposure to mixtures of 1,3-butadiene and styrene

1,3-Butadiene and styrene are oxidized, in part, by cytochrome P450 2E1 and have been shown to metabolically interact in rodents exposed by inhalation to mixtures of both compounds. Because the reactive metabolites of butadiene and styrene are thought to be responsible for the toxicity of each compound, metabolic interactions may alter the response in animals exposed to mixtures of butadiene and styrene compared with the response in animals exposed to butadiene alone or styrene alone. The purpose of this study was to quantitate alterations in genotoxicity and cytotoxicity in male B6C3F1 mice exposed to mixtures of butadiene and styrene. Male B6C3F1 mice were exposed to 6.25, 62.5, 200, or 625 ppm butadiene alone, 50 ppm styrene alone, or mixtures of 6.25, 62.5, 200, or 625 ppm butadiene and 50 ppm styrene. Genotoxicity was assessed by quantitating the frequency of micronucleated polychromatic erythrocytes in bone marrow. Cytotoxicity was assessed by counting total spleen and thymus cells and by quantitating the frequency of polychromatic erythrocytes in the peripheral blood. Butadiene and mixtures of butadiene and styrene were genotoxic in mice, as shown by a significant increase in the frequency of micronucleated polychromatic erythrocytes. The increased frequency following exposure to mixtures of butadiene and styrene was not significantly different compared with the frequency following exposure to butadiene alone. Styrene and mixtures of butadiene and styrene were cytotoxic in mice, as shown by significantly decreased number of spleen cells. Exposure to mixtures of butadiene and styrene with butadiene concentrations of 62.5 or 625 ppm significantly reduced the number of thymus cells. Exposure to 200 ppm or 625 ppm butadiene alone, or to mixtures of 200 ppm or 625 ppm butadiene and 50 ppm styrene, significantly reduced the frequency of polychromatic erythrocytes in the peripheral blood. The results of the study demonstrate that exposure to mixtures of butadiene and styrene does not reduce the respective genotoxicity of butadiene or cytotoxicity of styrene. Environ. Mol. Mutagen. 29: 335–345, 1997. © 1997 Wiley-Liss, Inc.